Factor Receptor Substrate Research Articles

Consistent Amplification of FRS2 and MDM2 in Low-grade Osteosarcoma: A Genetic Study of 22 Cases With Clinicopathologic Analysis.

Low-grade osteosarcoma (LGOS) encompasses low-grade central osteosarcoma (LGCOS) and parosteal osteosarcoma (POS). LGOSs are characterized by a supernumerary ring and giant rod chromosomes containing the 12q13-15 amplicon. The fibroblast growth factor receptor substrate 2 (FRS2) gene is located close to MDM2 and CDK4. Recent studies identified consistent amplification of FRS2 gene in atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma. The aim of this study was to evaluate the frequency of FRS2 amplification and its relationship with the clinicopathologic features of LGOSs. The amplification of FRS2 and MDM2 genes were analyzed by fluorescence in situ hybridization using 22 LGOSs (3 LGCOSs, 14 classic POSs, and 5 dedifferentiated POSs) and 85 control samples of bone and soft tissue. The clinicopathologic features of the 22 LGOSs were described. Amplification of FRS2 was detected in 21/22 (95%) of the LGOSs, including 3 (100%) LGCOSs and 18 (95%) POSs. All 22 LGOSs showed MDM2 amplification (100%). The only MDM2/FRS2 LGOS was dedifferentiated POS (the dedifferentiated component was conventional osteosarcoma). In the control group, all of the atypical lipomatous tumor/well-differentiated liposarcoma/dedifferentiated liposarcomas (DDLs) (10/10, 100%) were FRS2-amplified, whereas the remaining 75 control cases were FRS2-nonamplified. These findings indicate that the FRS2 gene is consistently amplified in classic and dedifferentiated LGOSs but not in their histologic mimics. These results offer another avenue for investigating the biology of LGOSs. Whether FRS2-nonamplified tumors exhibit unusual clinicopathologic features needs further investigation. Some so-called "high-grade osteosarcomas harboring 12q13-15 amplification" may be unrecognized dedifferentiated LGOSs.

Fibroblast growth factor receptor-1 is essential for regulation of mitochondrial biogenesis via control of microRNA let-7b

Objective To explore the role of fibroblast growth factor receptor (FGFR) 1 in endothelial homeostasis via an induction of microRNA let-7s, with effects on AcSDKP(N-acetyl-seryl-aspartyl-lysyl-proline) and associated mitochondrial biogenesis. Methods Blocking FGFR1 signaling pathway, Western blot and immunofluorescence staining were used to measure mitochondrial fusion ( mitofusin-2, MFN2; optic atrophy protein 1, OPA1) and fission (dynamin-related protein-1, DRP1) proteins and mitochondrial biogenesis by MitoTraker Green. Also real-time quantitative PCR(qPCR) was performed to test microRNA let-7′ expression. Results FGFR1 signaling pathway was critical for AcSDKP maintaining mitochondrial biogenesis through induction of microRNA let-7b. In endothelial cells, the AcSDKP restored the triple[TGF-β2, interleukin (IL)-1β, tumor necrosis factor (TNF)-α]-suppressed microRNA let-7b-5p expression and associated with mitochondrial biogenesis. Such effect of AcSDKP was lost in either fibroblast growth factor receptor substrate 2 (FRS2) siRNA or neutralizing FGFR1 treated-cells. Similarly, AcSDKP lost its effect on mitochondrial biogenesis in microRNA let-7b-5p inhibitor-treated-cells. In addition, microRNA let-7b-5p mimic reversed the FRS2 siRNA-suppressed mitochondrial biogenesis in endothelial cells. Conclusion These findings demonstrated that FGFR1 is critical for maintaining mitochondrial biogenesis through control of microRNA let-7b-5p in endothelial cells. (Chin J Endocrinol Metab, 2018, 34: 404-409) Key words: FGFR1; FRS2; Mitochondrial biogenesis; MicroRNA let-7b-5p

FGFR1 is essential for N-acetyl-seryl-aspartyl-lysyl-proline regulation of mitochondrial dynamics by upregulating microRNA let-7b-5p

Fibroblast growth factor receptor (FGFR) 1 plays a key role in endothelial homeostasis by inducing microRNA (miR) let-7. Our previous paper showed that anti-fibrotic effects of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) were associated with restoring diabetes-suppressed expression of FGFR1 and miR let-7, the key contributor of mitochondrial biogenesis, which is regulated by mitochondrial membrane GTPase proteins (MFN2 and OPA1). Here, we found that the FGFR1 signaling pathway was critical for AcSDKP in maintaining endothelial mitochondrial biogenesis through induction of miR let-7b-5p. In endothelial cells, AcSDKP restored the triple cytokines (TGF-β2, interleukin-1β, tumor necrosis factor-α)-suppressed miR let-7b-5p and protein levels of the mitochondrial membrane GTPase. This effect of AcSDKP was lost with either fibroblast growth factor receptor substrate 2 (FRS2) siRNA or neutralizing FGFR1-treated cells. Similarly, AcSDKP had no effect on the miR let-7b-5p inhibitor-suppressed GTPase levels in endothelial cells. In addition, a miR let-7b-5p mimic restored the levels of FRS2 siRNA-reduced GTPases in endothelial cells. These findings were also confirmed using MitoTracker Green and an immunofluorescence assay. Our results demonstrated that the AcSDKP-FGFR1 signaling pathway is critical for maintaining mitochondrial dynamics by control of miR let-7b-5p in endothelial cells.

MiR-339 inhibits proliferation of pulmonary artery smooth muscle cell by targeting FGF signaling.

Pulmonary artery hypertension (PAH) is a fatal disorder. Recent studies suggest that microRNA (miRNA) plays an important role in regulating proliferation of pulmonary artery smooth muscle cells (PASMC), which underlies the pathology of PAH. However, the exact mechanism of action of miRNAs remains elusive. In this study, we found that miR‐339 was highly expressed in the cardiovascular system and was downregulated by a group of cytokines and growth factors, especially PDGF‐BB and FGF2. Functional analyses revealed that miR‐339 can inhibit proliferation of PASMC. Also, miR‐339 inhibited FGF2‐induced proliferation, but had no effect on proliferation induced by PDGF‐BB. The fibroblast growth factor receptor substrate 2 (FRS2) was identified as a potential direct target of miR‐339. Consistent with the actions of miR‐339, knockdown of FRS2 only inhibited FGF2‐ but not PDGF‐BB‐induced proliferation of PASMC. In addition, our results showed that inhibition of ERK and PI3K abrogated the downregulation of miR‐339 induced by PDGF‐BB. Finally, miR‐339 expression was found to be decreased in the pulmonary arteries of rats with MCT‐induced PAH. Our study is the first report on the biological role of miR‐339 in regulating proliferation of PASMC by targeting FGF signaling, providing new mechanistic insights into PASMC proliferation and pathogenesis of PAH.

Identification of miRNA and genes involving in osteosarcoma by comprehensive analysis of microRNA and copy number variation data.

The aim of the present study was to understand the molecular mechanisms of osteosarcoma by comprehensive analysis of microRNA (miRNA/miR) and copy number variation (CNV) microarray data. Microarray data (GSE65071 and GSE33153) were downloaded from the Gene Expression Omnibus. In GSE65071, differentially expressed miRNAs between the osteosarcoma and control groups were calculated by the Limma package. Target genes of differentially expressed miRNAs were identified by the starBase database. For GSE33153, PennCNV software was used to perform the copy number variation (CNV) analysis. Overlapping of the genes in CNV regions and the target genes of differentially expressed miRNAs were used to construct miRNA-gene regulatory network using the starBase database. A total of 149 differentially expressed miRNAs, including 13 downregulated and 136 upregulated, were identified. In the GSE33153 dataset, 987 CNV regions involving in 3,635 genes were identified. In total, 761 overlapping genes in 987 CNV regions and in the genes in 7,313 miRNA-gene pairs were obtained. miRNAs (hsa-miR-27a-3p, hsa-miR-124-3p, hsa-miR-9-5p, hsa-miR-182-5p, hsa-miR-26a-5p) and the genes [Fibroblast growth factor receptor substrate 2 (FRS2), coronin 1C (CORO1C), forkhead box P1 (FOXP1), cytoplasmic polyadenylation element binding protein 4 (CPEB4) and glucocorticoid induced 1 (GLCCI1)] with the highest degrees of association with osteosarcoma development were identified. Hsa-miR-27a-3p, hsa-miR-9-5p, hsa-miR-182-5p, FRS2, CORO1C, FOXP1 and CPEB4 may be involved in osteosarcoma pathogenesis, and development.

Expression of signaling adaptor proteins predicts poor prognosis in pancreatic ductal adenocarcinoma

BackgroundAdaptor proteins bridge the gap between cell surface receptors and their downstream signaling elements. The clinicopathological and prognostic values of adaptor proteins remain poorly understood. The purpose of the present study was to explore the expression and prognostic value of three adaptor proteins: GRB2-associated binding protein 2 (GAB2), CRK-like protein (CRKL) and fibroblast growth factor receptor substrate 2 (FRS2) in pancreatic ductal adenocarcinoma (PDAC).MethodsThe expression of GAB2, CRKL, and FRS2 in 77 formalin fixed paraffin embedded (FFPE) samples from 77 PDAC patients, along with three paired fresh PDAC and matched normal tissues from 3 PDAC patients was analyzed by immunohistochemistry and western blot, respectively. The association between the expression of the three proteins and the clinicopathological factors of PDAC was assessed by χ2 test. The correlation between the expression levels of the three proteins was analyzed by Spearman rank correlation analyses; Kaplan-Meier survival analyses were also performed.ResultsIHC was successful in 75, 76, and 77 cases for GAB2, CRKL, and FRS2, respectively. Of which, the positive rate of GAB2, CRKL, and FRS2 protein expression was 40.00% (30/75), 53.95% (41/76) and 35.06% (27/77), respectively. The positive rate of GAB2, CRKL and FRS2 co-expression was 16.88% (13/77). Though there was no association between GAB2 expression, CRKL expression, FRS2 expression, GAB2/CRKL/FRS2 co-expression and the clinicopathological parameters of PDAC, positive correlations were observed between the expressions of the three proteins. Further, univariate survival analysis showed that positive expression of GAB2, CRKL and FRS2 and co-expression of GAB2/CRKL/FRS2 of PDAC predicted poor clinical outcomes, and multivariate survival analysis suggested that positive expression of GAB2 and positive co-expression of GAB2/CRKL/FRS2 were independent prognostic factors for disease-free survival (DFS) and overall survival (OS), respectively.ConclusionIn conclusion, GAB2, CRKL, and FRS2 may be potential prognosticators and therapeutic targets for PDAC patients.

Androgen suppresses protein kinase D1 expression through fibroblast growth factor receptor substrate 2 in prostate cancer cells.

In prostate cancer, androgen/androgen receptor (AR) and their downstream targets play key roles in all stages of disease progression. The protein kinase D (PKD) family, particularly PKD1, has been implicated in prostate cancer biology. Here, we examined the cross-regulation of PKD1 by androgen signaling in prostate cancer cells. Our data showed that the transcription of PKD1 was repressed by androgen in androgen-sensitive prostate cancer cells. Steroid depletion caused up regulation of PKD1 transcript and protein, an effect that was reversed by the AR agonist R1881 in a time- and concentration-dependent manner, thus identifying PKD1 as a novel androgen-repressed gene. Kinetic analysis indicated that the repression of PKD1 by androgen required the induction of a repressor protein. Furthermore, inhibition or knockdown of AR reversed AR agonist-induced PKD1 repression, indicating that AR was required for the suppression of PKD1 expression by androgen. Downstream of AR, we identified fibroblast growth factor receptor substrate 2 (FRS2) and its downstream MEK/ERK pathway as mediators of androgen-induced PKD1 repression. In summary, PKD1 was identified as a novel androgen-suppressed gene and could be downregulated by androgen through a novel AR/FRS2/MEK/ERK pathway. The upregulation of prosurvival PKD1 by anti-androgens may contribute to therapeutic resistance in prostate cancer treatment.

Clinical Aspects of Glucose Metabolism and Chronic Disease.

The burden of chronic disease is an emerging world health problem. Advances made in the treatment of individual disease states often fail to consider multimorbidity patterns in clinical research models. Adjusting for age as a confounder ignores its contribution as a powerful risk factor for most chronic diseases. Sarcopenia is an age-related loss of skeletal muscle mass, which is accelerated by chronic inflammation and its resulting cascade of cytokines. Skeletal muscle loss results in insulin resistance, hyperglycemia, and altered mitochondrial glucose signaling pathways. Vascular disease in the brain may alter blood-brain barrier function, allowing transport of substances into the brain which adversely affect the "astrocyte-centric" subunit. Neurogenesis that provides neuronal plasticity is impaired in the diabetic brain, while insulin resistance markers such as insulin-like growth factor (IGF-1) and insulin receptor substrate (IRS-1) are associated with poor cognitive performance. Advanced glycation end products generated by chronic hyperglycemia are found in postmortem AD brain. Intranasal insulin administration, a preferential route for CNS delivery, improved cognitive function in healthy adults, without affecting circulating levels of insulin or glucose. Exercise has demonstrated a neuroprotective effect through induction of antioxidative enzymes, neurotrophic, and vascular endothelial growth factors. Sarcopenia appears to be a dynamic process and is potentially reversible with attention to nutrition and cardiovascular fitness. Early detection and intervention may slow the progression of multimortality disease states and should be a focus of worldwide health systems.

Coupling an EML4-ALK-centric interactome with RNA interference identifies sensitizers to ALK inhibitors.

Patients with lung cancers harboring anaplastic lymphoma kinase (ALK) gene fusions benefit from treatment with ALK inhibitors, but acquired resistance inevitably arises. A better understanding of proximal ALK signaling mechanisms may identify sensitizers to ALK inhibitors that disrupt the balance between prosurvival and proapoptotic effector signals. Using affinity purification coupled with mass spectrometry in an ALK fusion lung cancer cell line (H3122), we generated an ALK signaling network and investigated signaling activity using tyrosine phosphoproteomics. We identified a network of 464 proteins composed of subnetworks with differential response to ALK inhibitors. A small hairpin RNA screen targeting 407 proteins in this network revealed 64 and 9 proteins that when knocked down sensitized cells to crizotinib and alectinib, respectively. Among these, knocking down fibroblast growth factor receptor substrate 2 (FRS2) or coiled-coil and C2 domain-containing protein 1A (CC2D1A), both scaffolding proteins, sensitized multiple ALK fusion cell lines to the ALK inhibitors crizotinib and alectinib. Collectively, our data set provides a resource that enhances our understanding of signaling and drug resistance networks consequent to ALK fusions and identifies potential targets to improve the efficacy of ALK inhibitors in patients.

Fibroblast Growth Factor Receptor-Dependent and -Independent Paracrine Signaling by Sunitinib-Resistant Renal Cell Carcinoma.

Antiangiogenic therapies, such as sunitinib, have revolutionized renal cell carcinoma (RCC) treatment. However, a precarious understanding of how resistance emerges and a lack of tractable experimental systems hinder progress. We evaluated the potential of primary RCC cultures (derived from tumors and tumor grafts) to signal to endothelial cells (EC) and fibroblasts in vitro and to stimulate angiogenesis ex vivo in chorioallantoic membrane (CAM) assays. From 65 patients, 27 primary cultures, including several from patients with sunitinib-resistant RCC, were established. RCC cells supported EC survival in coculture assays and induced angiogenesis in CAM assays. RCC-induced EC survival was sensitive to sunitinib in half of the tumors and was refractory in tumors from resistant patients. Sunitinib sensitivity correlated with vascular endothelial growth factor (VEGF) production. RCC induced paracrine extracellular signal-regulated kinase (ERK) activation in EC which was inhibited by sunitinib in sensitive but not in resistant tumors. As determined by fibroblast growth factor receptor substrate 2 (FRS2) phosphorylation in fibroblasts, RCC broadly induced low-level fibroblast growth factor receptor (FGFR) signaling. Whereas ERK activation in EC was uniformly inhibited by combined VEGF/platelet-derived growth factor (PDGF)/FGF receptor inhibitors, paracrine ERK activation in fibroblasts was blocked in only a fraction of tumors. Our data show that RCC activates EC through VEGF-dependent and -independent pathways, that sunitinib sensitivity correlates with VEGF-mediated ERK activation, and that combined inhibition of VEGF/PDGF/FGF receptors is sufficient to inhibit mitogenic signaling in EC but not in fibroblasts.

Identification of Oncogenic and Drug-Sensitizing Mutations in the Extracellular Domain of FGFR2.

The discovery of oncogenic driver mutations and the subsequent developments in targeted therapies have led to improved outcomes for subsets of lung cancer patients. The identification of additional oncogenic and drug-sensitive alterations may similarly lead to new therapeutic approaches for lung cancer. We identify and characterize novel FGFR2 extracellular domain insertion mutations and demonstrate that they are both oncogenic and sensitive to inhibition by FGFR kinase inhibitors. We demonstrate that the mechanism of FGFR2 activation and subsequent transformation is mediated by ligand-independent dimerization and activation of FGFR2 kinase activity. Both FGFR2-mutant forms are predominantly located in the endoplasmic reticulum and Golgi but nevertheless can activate downstream signaling pathways through their interactions with fibroblast growth factor receptor substrate 2 (FRS2). Our findings provide a rationale for therapeutically targeting this unique subset of FGFR2-mutant cancers as well as insight into their oncogenic mechanisms.

The Role of Fibroblast Growth Factor Receptor Substrate 2 (FRS2) in the Regulation of Two Activity Levels of the Components of the Extracellular Signal-Regulated Kinase (ERK) Pathway in the Mouse Epididymis1

The components of the extracellular signal-regulated kinase (ERK) pathway are involved in the regulation of epididymal cellular processes. Interestingly, our previous studies showed that there are two different activity levels of the ERK pathway components in the epididymal epithelium: a basal level in most regions and a higher level in the differentiated initial segment (IS). In this study we analyzed the role of fibroblast growth factor receptor substrate 2 (FRS2) in the regulation of these two levels. Two mouse models were generated. In the first model, Frs2 was deleted from epithelial cells of most epididymal regions except for the IS from the embryonic period onward. Loss of Frs2 dampened the basal activity level of the ERK pathway components, which resulted in an increase in apoptosis along the epididymal duct. This was observed during the period when FRS2 expression level was highest in wild-type epididymides. In the second model, Frs2 was deleted from the proximal epididymal epithelium from Postnatal Day 17 onward. Most of the epididymides in this model exhibited normal morphology. Loss of Frs2 in these epididymides did not affect the high activity level of the ERK pathway components in the IS. However, a subgroup of epididymides in the second model showed increased apoptosis which resulted in an abnormally shaped proximal region or development of granulomas. Therefore, data from these two models showed that FRS2 played different roles in the regulation of two activity levels of the ERK pathway components in the epididymis.

Involvement of β1-Integrin Up-regulation in Basic Fibroblast Growth Factor- and Epidermal Growth Factor-induced Proliferation of Mouse Neuroepithelial Cells

In neural stem cells, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promote cell proliferation and self-renewal. In the bFGF- and EGF-responsive neural stem cells, beta1-integrin also plays important roles in crucial cellular processes, including proliferation, migration, and apoptosis. The cross-talk of the signaling pathways mediated by these growth factors and beta1-integrin, however, has not been fully elucidated. Here we report a novel molecular mechanism through which bFGF or EGF promotes the proliferation of mouse neuroepithelial cells (NECs). In the NECs, total beta1-integrin expression levels and proliferation were dose-dependently increased by bFGF but not by EGF. EGF rather than bFGF strongly induced the increase of beta1-integrin localization on the NEC surface. bFGF- and EGF-induced beta1-integrin up-regulation and proliferation were inhibited after treatment with a mitogen-activated protein kinase kinase inhibitor, U0126, which indicates the dependence on the mitogen-activated protein kinase pathway. Involvement of beta1-integrin in bFGF- and EGF-induced proliferation was confirmed by the finding that NEC proliferation and adhesion to fibronectin-coated dishes were inhibited by knockdown of beta1-integrin using small interfering RNA. On the other hand, apoptosis was induced in NECs treated with RGD peptide, a small beta1-integrin inhibitor peptide with the Arg-Gly-Asp motif, but it was independent of beta1-integrin expression levels. Those results suggest that regulation of beta1-integrin expression/localization is involved in cellular processes, such as proliferation, induced by bFGF and EGF in NECs. The mechanism underlying the proliferation through beta1-integrin would not be expected to be completely identical, however, for bFGF and EGF.

Targeting Fibroblast Growth Factor Receptors Blocks PI3K/AKT Signaling, Induces Apoptosis, and Impairs Mammary Tumor Outgrowth and Metastasis

Members of the fibroblast growth factor receptor (FGFR) family have essential roles in normal physiology and in cancer where they control diverse processes. FGFRs have been associated with breast cancer development. Thus, models to study the role of FGFR in breast cancer and their targeting potential are important. We present an in vitro and in vivo analysis of FGFRs in the breast cancer model cell lines 67NR and 4T1. We show that both tumor cell lines coexpress FGFRs and ligands and display autocrine FGFR signaling activity. Fibroblast growth factor receptor substrate 2 (FRS2), a downstream mediator of FGFR, is constitutively tyrosine phosphorylated and multiple signaling pathways are active. Treatment of 67NR and 4T1 cultures with TKI258, an FGFR tyrosine kinase inhibitor (TKI), caused a rapid decrease in FRS2 phosphorylation; decreased the activity of extracellular signal-regulated kinase 1/2 (ERK1/2), AKT, and phospholipase Cgamma; and blocked proliferation of both tumor lines. Furthermore, TKI258 induced 4T1 apoptotic cell death via blockade of the phosphoinositide 3-kinase/AKT pathway. In vivo, one dose of TKI258 rapidly lowered FRS2 phosphorylation and ERK1/2 and AKT activity in mammary tumors. Long-term TKI258 treatment of 4T1 tumor- and 67NR tumor-bearing mice had a significant effect on primary tumor outgrowth and 4T1 tumor-induced lung metastases. A microarray analysis was carried out to identify targets with roles in TKI258 antitumor activity and potential prognostic markers in human breast tumors. Of interest are the downregulated matrix metalloproteases (MMP), in particular MMP9, which is essential for metastatic spread of 4T1 tumors.

Abstract 3380: The serine protease inhibitor PN-1 induces activation of FGFR-mediated signaling

Abstract We are studying the role of PN-1 (Protease Nexin-1) in breast cancer. PN-1 is a serine protease inhibitor (serpin) that blocks a broad spectrum of proteases including among others tPA, uPA and thrombin. Once bound to a target protease, the PN-1/protease complexes bind with high affinity to Low Density Lipoprotein Receptor related Protein (LRP-1). A bioinformatic analysis revealed that PN-1 is overexpressed in a significant proportion of human tumors from different origins. Moreover, PN-1 expression levels correlate with markers of poor prognosis in breast cancer. PN-1 is also expressed in breast tumor cell lines and we recently showed that PN-1 is required by the malignant 4T1 mammary cancer cell line to metastasize to the lungs. Indeed, when mammary fat pads of Balb/c mice were injected with PN-1-knock down (KD)-4T1 cells, primary mammary tumors developed and these grew similarly as control 4T1 cells. However, the PN-1 KD tumors displayed a significant decrease in their potential to form lung metastasis, in comparison to control tumors. The mechanism by which complexes of serpin/protease mediate their effects in cancer cells is still largely unknown. Thus, our current studies are aimed at deciphering how the protease/PN-1 complex impacts on intracellular signaling in order to promote metastasis. Using the PN-1 negative 168FARN mammary tumor cell line, we have found that PN-1/protease complexes bind LRP-1 receptor and induce MMP-9 expression via Erk1/2 pathway activation. Importantly, the PN-1-driven regulation of MMP-9 expression was also demonstrated to be central to PN-1's prometastatic effect in 4T1 cells. Here by the use of Western Blot and specific inhibitors, we show that PN-1/protease complex treatment of fibroblast growth factor receptor (FGFR)-expressing tumor cells induces Erk signaling. Furthermore, fibroblast growth factor receptor substrate 2 (FRS2), a lipid anchored docking protein that plays a crucial role in mediating FGFR and tropomyosin receptor kinase (Trk)-induced signaling, was also found to be phosphorylated upon PN-1/protease complex addition. Preincubation of the cells with the FGFR inhibitor, TKI 258, abrogated phosphorylation of FRS2, Erk and Shc upon PN-1/protease complex treatment. In contrast, inhibitors of EGFR or PDGR did not have any effect on PN-1-induced phosphorylation of these proteins. This suggests that the PN-1/protease complex via binding to LRP-1 may transactivate FGFR and potentially other receptors known to activate mitogenic signaling in cancer cells. Our results are in agreement with a recent study showing that other ligands for LRP-1, (e.g, tPA and α2-macroglobulin), could transactivate TrkA receptor. Thus, PN-1 may also transactivate tyrosine kinase receptors. Hence, these results suggest that PN-1/protease complexes might be considered as a new target in cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3380.

The FRS2 family of docking/scaffolding adaptor proteins as therapeutic targets of cancer treatment

Background: There are two members – FRS2α and FRS2β – in the fibroblast growth factor receptor substrate 2 (FRS2) family of docking/scaffolding adaptor proteins. These proteins function downstream of certain kinds of receptor tyrosine kinases (RTKs) that are important for tumorigenesis. FRS2α acts as a control centre for fibroblast growth factor receptor signalling and encourages tumorigenesis, while FRS2β regulates EGFR signalling negatively, and might have a tumour suppressive role. Therefore, both proteins could be good therapeutic targets for the treatment of cancer. Objective: To examine the physiological and pathological roles of FRS2, especially in cancer, and describe their potential value as therapeutic targets. Methods: A review of relevant literature. Results/conclusions: Although it is still difficult to develop small compounds to modify functions of FRS2 adaptor proteins, such compounds may be useful as the next generation of molecular targeting drugs. Combination therapy with RTK-targeting drugs and FRS2-targeting drugs may be useful for cancer treatment in the near future.

Interaction of Mint2 with TrkA Is Involved in Regulation of Nerve Growth Factor-induced Neurite Outgrowth

TrkA receptor signaling is essential for nerve growth factor (NGF)-induced survival and differentiation of sensory neurons. To identify possible effectors or regulators of TrkA signaling, yeast two-hybrid screening was performed using the intracellular domain of TrkA as bait. We identified muc18-1-interacting protein 2 (Mint2) as a novel TrkA-binding protein and found that the phosphotyrosine binding domain of Mint2 interacted with TrkA in a phosphorylation- and ligand-independent fashion. Coimmunoprecipitation assays showed that endogenous TrkA interacted with Mint2 in rat tissue homogenates, and immunohistochemical evidence revealed that Mint2 and TrkA colocalized in rat dorsal root ganglion neurons. Furthermore, Mint2 overexpression inhibited NGF-induced neurite outgrowth in both PC12 and cultured dorsal root ganglion neurons, whereas inhibition of Mint2 expression by RNA interference facilitated NGF-induced neurite outgrowth. Moreover, Mint2 was found to promote the retention of TrkA in the Golgi apparatus and inhibit its surface sorting. Taken together, our data provide evidence that Mint2 is a novel TrkA-regulating protein that affects NGF-induced neurite outgrowth, possibly through a mechanism involving retention of TrkA in the Golgi apparatus.

Prominent expression of FRS2β protein in neural cells and its association with intracellular vesicles

The fibroblast growth factor receptor substrate (FRS)-2 protein family comprises FRS2α, a well-known central mediator for fibroblast growth factor signaling, and FRS2β, whose endogenous expression pattern and function are not yet defined. Immunohistochemical analysis revealed that expression of FRS2β was restricted to neural tissues and it colocalized with Tuj1, a neuronal marker. There are two distinct patterns of FRS2β expression in neural cells: punctate and cup/ring-shaped; moreover, some particles colocalized with lysosomes. Stimulation with brain-derived neurotrophic factor (BDNF) enhanced FRS2β phosphorylation and the cup/ring-shaped pattern. These results suggest a probable role of FRS2β in the intracellular degradation systems of neural cells, which involves lysosomes.

Src kinase modulates the activation, transport and signalling dynamics of fibroblast growth factor receptors

The non-receptor tyrosine kinase Src is recruited to activated fibroblast growth factor receptor (FGFR) complexes through the adaptor protein factor receptor substrate 2 (FRS2). Here, we show that Src kinase activity has a crucial role in the regulation of FGFR1 signalling dynamics. Following receptor activation by ligand binding, activated Src is colocalized with activated FGFR1 at the plasma membrane. This localization requires both active Src and FGFR1 kinases, which are inter-dependent. Internalization of activated FGFR1 is associated with release from complexes containing activated Src. Src-mediated transport and subsequent activation of FGFR1 require both RhoB endosomes and an intact actin cytoskeleton. Chemical and genetic inhibition studies showed strikingly different requirements for Src family kinases in FGFR1-mediated signalling; activation of the phosphoinositide-3 kinase-Akt pathway is severely attenuated, whereas activation of the extracellular signal-regulated kinase pathway is delayed in its initial phase and fails to attenuate.

Keratinocyte Growth Factor Receptor Ligands Target the Receptor to Different Intracellular Pathways

The keratinocyte growth factor receptor (KGFR)/fibroblast growth factor receptor 2b is activated by high-affinity-specific interaction with two different ligands, keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)7 and FGF10/KGF2, which are characterized by an opposite requirement of heparan sulfate proteoglycans and heparin for binding to the receptor. We investigated here the possible different endocytic trafficking of KGFR, induced by the two ligands. Immunofluorescence and immunoelectron microscopy analysis showed that KGFR internalization triggered by either KGF or FGF10 occurs through clathrin-coated pits. Immunofluorescence confocal microscopy using endocytic markers as well as tumor susceptibility gene 101 (TSG101) silencing demonstrated that KGF drives KGFR to the degradative pathway, while FGF10 targets the receptor to the recycling endosomes. Biochemical analysis showed that KGFR is ubiquitinated and degraded after KGF treatment but not after FGF10 treatment, and that the alternative fate of KGFR might depend on the different ability of the receptor to phosphorylate the fibroblast growth factor receptor substrate 2 (FRS2) substrate and to recruit the ubiquitin ligase c-Cbl. The recycling endocytic pathway followed by KGFR upon FGF10 stimulation correlates with the higher mitogenic activity exerted by this ligand on epithelial cells compared with KGF, suggesting that the two ligands may play different functional roles through the regulation of the receptor endocytic transport.