FOXA1 Promotes Tumor Progression in Prostate Cancer and Represents a Novel Hallmark of Castration-Resistant Prostate Cancer

Forkhead box protein A1 (FOXA1) modulates the transactivation of steroid hormone receptors and thus may influence tumor growth and hormone responsiveness in prostate cancer. We therefore investigated the correlation of FOXA1 expression with clinical parameters, prostate-specific antigen (PSA) relapse-free survival, and hormone receptor expression in a large cohort of prostate cancer patients at different disease stages. FOXA1 expression did not differ significantly between benign glands from the peripheral zone and primary peripheral zone prostate carcinomas. However, FOXA1 was overexpressed in metastases and particularly in castration-resistant cases, but was expressed at lower levels in both normal and neoplastic transitional zone tissues. FOXA1 levels correlated with higher pT stages and Gleason scores, as well as with androgen (AR) and estrogen receptor expression. Moreover, FOXA1 overexpression was associated with faster biochemical disease progression, which was pronounced in patients with low AR levels. Finally, siRNA-based knockdown of FOXA1 induced decreased cell proliferation and migration. Moreover, in vitro tumorigenicity was inducible by ARs only in the presence of FOXA1, substantiating a functional cooperation between FOXA1 and AR. In conclusion, FOXA1 expression is associated with tumor progression, dedifferentiation of prostate cancer cells, and poorer prognosis, as well as with cellular proliferation and migration and with AR signaling. These findings suggest FOXA1 overexpression as a novel mechanism inducing castration resistance in prostate cancer.

Interaction of the receptor FGFRL1 with the negative regulator Spred1

FGFRL1 is a member of the fibroblast growth factor receptor family. It plays an essential role during branching morphogenesis of the metanephric kidneys, as mice with a targeted deletion of the Fgfrl1 gene show severe kidney dysplasia. Here we used the yeast two-hybrid system to demonstrate that FGFRL1 binds with its C-terminal, histidine-rich domain to Spred1 and to other proteins of the Sprouty/Spred family. Members of this family are known to act as negative regulators of the Ras/Raf/Erk signaling pathway. Truncation experiments further showed that FGFRL1 interacts with the SPR domain of Spred1, a domain that is shared by all members of the Sprouty/Spred family. The interaction could be verified by coprecipitation of the interaction partners from solution and by codistribution at the cell membrane of COS1 and HEK293 cells. Interestingly, Spred1 increased the retention time of FGFRL1 at the plasma membrane where the receptor might interact with ligands. FGFRL1 and members of the Sprouty/Spred family belong to the FGF synexpression group, which also includes FGF3, FGF8, Sef and Isthmin. It is conceivable that FGFRL1, Sef and some Sprouty/Spred proteins work in concert to control growth factor signaling during branching morphogenesis of the kidneys and other organs.

RNA interference screening identifies a novel role for autocrine fibroblast growth factor signaling in neuroblastoma chemoresistance

Chemotherapeutic drug resistance is one of the major causes for treatment failure in high-risk neuroblastoma (NB), the most common extra cranial solid tumor in children. Poor prognosis is typically associated with MYCN amplification. Here, we utilized a loss-of-function kinome-wide RNA interference screen to identify genes that cause cisplatin sensitization. We identified fibroblast growth factor receptor 2 (FGFR2) as an important determinant of cisplatin resistance. Pharmacological inhibition of FGFR2 confirmed the importance of this kinase in NB chemoresistance. Silencing of FGFR2 sensitized NB cells to cisplatin-induced apoptosis, which was regulated by the downregulation of the anti-apoptotic proteins BCL2 and BCLX(L). Mechanistically, FGFR2 was shown to activate protein kinase C-δ to induce BCL2 expression. FGFR2, as well as the ligand fibroblast growth factor-2, were consistently expressed in primary NB and NB cell lines, indicating the presence of an autocrine loop. Expression analysis revealed that FGFR2 correlates with MYCN amplification and with advanced stage disease, demonstrating the clinical relevance of FGFR2 in NB. These findings suggest a novel role for FGFR2 in chemoresistance and provide a rational to combine pharmacological inhibitors against FGFR2 with chemotherapeutic agents for the treatment of NB.Oncogene advance online publication, 1 October 2012; doi:10.1038/onc.2012.416.

Phosphatase and tensin homolog regulates stability and activity of EphB1 receptor

Deregulation of receptor tyrosine kinases (RTKs) is linked to a broad range of cancers, stressing the necessity of studying their regulatory pathways. We and others demonstrated previously that c-Cbl is necessary for the lysosomal degradation of erythropoietin-producing hepatocellular B1 (EphB1) carcinoma and epidermal growth factor receptor (EGFR) RTKs. Moreover, the tumor suppressor phosphatase and tensin homolog (PTEN) was shown to modulate c-Cbl-dependent EGFR degradation. We therefore investigated the involvement of PTEN in EphB1 signaling and degradation. We used PTEN mutants, PTEN, and NHERF1 small interfering RNA in CHO-EphB1 and SW480 cells endogenously expressing EphB1 to delineate EphB1-PTEN interactions. PTEN was constitutively associated with c-Cbl, protecting it from degradation. EphB1 stimulation triggered ∼50% serine-threonine PTEN dephosphorylation and PTEN-Cbl complex disruption, a process requiring PTEN protein phosphatase activity. Both proteins independently translocated to EphB1, with PTEN in association with the scaffold protein NHERF1. Biologically, PTEN lipid phosphatase activity impairs EphB1-dependent cell adhesion and chemotaxis. This study demonstrates for the first time in mammalian cells that the Eph receptor and PTEN associate and influence their signaling. Moreover, it contributes to the emerging concept that PTEN regulates expression of RTKs through modulation of their degradation. Finally, it reveals a new role for PTEN protein phosphatase activity involved in this process.

Inhibition of the AKT/mTOR and erbB pathways by gefitinib, perifosine and analogs of gonadotropin-releasing hormone I and II to overcome tamoxifen resistance in breast cancer cells

Endocrine resistance in breast cancer remains a major clinical problem and is caused by crosstalk mechanisms of growth factor receptor cascades, such as the erbB and PI3K/AKT pathways. The possibilities a single breast cancer cell has to achieve resistance are manifold. We developed a model of 4-hydroxy-tamoxifen (OHT)‑resistant human breast cancer cell lines and compared their different expression patterns, activation of growth factor receptor pathways and compared cells by genomic hybridization (CGH). We also tested a panel of selective inhibitors of the erbB and AKT/mTOR pathways to overcome OHT resistance. OHT‑resistant MCF-7-TR and T47D-TR cells showed increased expression of HER2 and activation of AKT. T47D-TR cells showed EGFR expression and activated MAPK (ERK-1/2), whereas in resistant MCF-7-TR cells activated AKT was due to loss of CTMP expression. CGH analyses revealed remarkable aberrations in resistant sublines, which were predominantly depletions. Gefitinib inhibited erbB signalling and restored OHT sensitivity in T47D-TR cells. The AKT inhibitor perifosine restored OHT sensitivity in MCF-7-TR cells. All cell lines showed expression of receptors for gonadotropin-releasing hormone (GnRH) I and II, and analogs of GnRH-I/II restored OHT sensitivity in both resistant cell lines by inhibition of erbB and AKT signalling. In conclusion, mechanisms to escape endocrine treatment in breast cancer share similarities in expression profiling but are based on substantially different genetic aberrations. Evaluation of activated mediators of growth factor receptor cascades is helpful to predict response to specific inhibitors. Expression of GnRH-I/II receptors provides multi-targeting treatment strategies.

Functional interaction of vascular endothelial-protein-tyrosine phosphatase with the angiopoietin receptor Tie-2

During development of the vertebrate vascular system essential signals are transduced via protein-tyrosine phosphorylation. Null-mutations of receptor-tyrosine kinase (RTK) genes expressed in endothelial cells (ECs) display early lethal vascular phenotypes. We aimed to identify endothelial protein-tyrosine phosphatases (PTPs), which should have similar importance in EC-biology. A murine receptor-type PTP was identified by a degenerated PCR cloning approach from endothelial cells (VE-PTP). By in situ hybridization this phosphatase was found to be specifically expressed in vascular ECs throughout mouse development. In experiments using GST-fusion proteins, as well as in transient transfections, trapping mutants of VE-PTP co-precipitated with the Angiopoietin receptor Tie-2, but not with the Vascular Endothelial Growth Factor receptor 2 (VEGFR-2/Flk-1). In addition, VE-PTP dephosphorylates Tie-2 but not VEGFR-2. We conclude that VE-PTP is a Tie-2 specific phosphatase expressed in ECs, and VE-PTP phosphatase activity serves to specifically modulate Angiopoietin/Tie-2 function. Based on its potential role as a regulator of blood vessel morphogenesis and maintainance, VE-PTP is a candidate gene for inherited vascular malformations similar to the Tie-2 gene.

Outside-in signaling through integrins and cadherins: a central mechanism to control epidermal growth and differentiation?

The process of epidermal renewal persists throughout the entire life of an organism. It begins when a keratinocyte progenitor leaves the stem cell compartment, undergoes a limited number of mitotic divisions, exits the cell cycle, and commits to terminal differentiation. At the end of this phase, the postmitotic keratinocytes detach from the basement membrane to build up the overlaying stratified epithelium. Although highly coordinated, this sequence of events is endowed with a remarkable versatility, which enables the quiescent keratinocyte to reintegrate into the cell cycle and become migratory when necessary, for example after wounding. It is this versatility that represents the Achilles heel of epithelial cells allowing for the development of severe pathologies. Over the past decade, compelling evidence has been provided that epithelial cancer cells achieve uncontrolled proliferation following hijacking of a "survival program" with PI3K/Akt and a "proliferation program" with growth factor receptor signaling at its core. Recent insights into adhesion receptor signaling now propose that integrins, but also cadherins, can centrally control these programs. It is suggested that the two types of adhesion receptors act as sensors to transmit extracellular stimuli in an outside-in mode, to inversely modulate epidermal growth factor receptor signaling and ensure cell survival. Hence, cell-matrix and cell-cell adhesion receptors likely play a more powerful and wide-ranging role than initially anticipated. This Perspective article discusses the relevance of this emerging field for epidermal growth and differentiation, which can be of importance for severe pathologies such as tumorigenesis and invasive metastasis, as well as psoriasis and Pemphigus vulgaris.

Nuclear localization of epidermal growth factor and epidermal growth factor receptors in human thyroid tissues

Epidermal growth factor (EGF) has widespread growth effects, and in some tissues proliferation is associated with the nuclear localization of EGF and epidermal growth factor receptor (EGFR). In the thyroid, EGF promotes growth but differs from thyrotropin (TSH) in inhibiting rather than stimulating functional parameters. We have therefore studied the occurrence and cellular distribution of EGF and EGFR in normal thyroid, in Graves' disease, where growth is mediated through the thyrotropin receptor (TSHR), and in a variety of human thyroid tumors. In the normal gland the staining was variable, but largely cytoplasmic, for both EGF and EGFR. In Graves' disease there was strong cytoplasmic staining for both EGF and EGFR, with frequent positive nuclei. Nuclear positivity for EGF and particularly for EGFR was also a feature of both follicular adenomas and follicular carcinomas. Interestingly, nuclear staining was almost absent in papillary carcinomas. These findings document for the first time the presence of nuclear EGF and EGFR in thyroid. Their predominant occurrence in tissues with increased growth (Graves' disease, follicular adenoma, and carcinoma) may indicate that nuclear EGF and EGFR play a role in growth regulation in these conditions. The absence of nuclear EGF and EGFR in papillary carcinomas would suggest that the role played by EGF in growth control differs between papillary carcinoma and follicular adenomas/carcinomas of the thyroid.

Regulation of fetal growth: consequences and impact of being born small

The first trimester of pregnancy is the time during which organogenesis takes place and tissue patterns and organ systems are established. In the second trimester the fetus undergoes major cellular adaptation and an increase in body size, and in the third trimester organ systems mature ready for extrauterine life. In addition, during that very last period of intrauterine life there is a significant increase in body weight. In contrast to the postnatal endocrine control of growth, where the principal hormones directly influencing growth are growth hormone (GH) and the insulin-like growth factors (IGFs) via the GH-IGF axis, fetal growth throughout gestation is constrained by maternal factors and placental function and is coordinated by growth factors. In general, growth disorders only become apparent postnatally, but they may well be related to fetal life. Thus, fetal growth always needs to be considered in the overall picture of human growth as well as in its metabolic development.

Experimental arthritis: effect on growth parameters and total skeletal calcium

Aim of the study was to investigate the possible mechanisms leading to stunted growth and osteoporosis in experimental arthritis. Fourty-two female rats of 7-8 weeks of age were randomly assigned to three groups of 14 animals each: (a) controls; (b) adjuvant-inoculated (AA); and (c) adjuvant-inoculated rats receiving 10 mg cyclosporin A (CsA) orally for 30 days. Biological parameters studied were: hindpaw swelling; vertebral length progression expressed as Delta increments between days 1 and 30 as a parameter of skeletal growth, and estimation of total skeletal mineral content by dual energy X-ray absorptiometry (n=10 each group) on day 30. Endocrine parameters measured were pulsatile release of growth hormone (rGH) on day 30 following jugular cannulation and measurement of insulin-like growth factor (IGF-1) in pooled plasma from rGH profiles. Results can be summarized as follows: Untreated AA rats exhibited local signs of inflammation in comparison with controls (hindpaw diameter 8.1-8.9 mm vs. 5.3-5.6 mm in controls). Treatment with CsA normalized this parameter (4.9-5.6 mm). Vertebral growth was significantly retarded in AA rats in comparison with controls (214+/-32 vs. 473+/-33 microm; p<0.001). Administration of CsA normalized vertebral size increment with a clear tendency to overgrowth (523+/-43 microm, n.s.). There was also a marked reduction in total skeletal mineral content in diseased (AA) rats as compared to controls (5.8+/-0.1 vs. 7.5+/-0.1g [OH-apatite]; p<0.001), and a moderate but significant increment above controls in the group receiving CsA (8.0+/-0.1 vs. 7.5+/-0.1g [OH-appatite]; p<0.04). Integrated rGH profiles exhibited a significant fall in arthritic rats and were completely restored to normal under CsA treatment. A trend toward higher rGH values was observed in the latter group (2908+/-554 in AA vs. 8317+/-1492 ng/ml/240 min in controls; p<0.001, and 10940+/-222 ng/ml/240 min, n.s. in the CsA group). There was a good correlation between skeletal growth and rGH pulsatility (r=0.81; p<0.001). IGF-1 followed a similar pattern (630+/-44 in AA vs. 752+/-30 ng/ml in controls; p<0.04, and 769+/-59 ng/ml in the CsA group, n.s. vs. controls). Thus, a clear tendency to skeletal overgrowth following treatment was observed in agreement with the hormonal data. It can therefore be concluded that, in experimental arthritis, attenuated GH-spiking and reduced circulating IGF-1 appear to be causally related to growth retardation, probably mimicking signs and symptoms observed in juvenile arthritis. Therapy with CsA is followed by normalization of hormonal and biological parameters accompanied by a catch up phenomenon in skeletal growth which is also observed clinically in juvenile arthritis. Generalized osteopenia is a prominent feature seemingly connected with the growth abnormalities as they parallel each other during the evolution of the disease and respond equally to therapy.

Associations with multiple pituitary hormone deficiency in patients with an ectopic posterior pituitary gland

INTRODUCTION: The presence of an ectopic posterior pituitary gland (EPP) on magnetic resonance imaging (MRI) is associated with hypopituitarism with one or more hormone deficiencies. We aimed to identify risk factors for having multiple pituitary hormone deficiency (MPHD) compared to isolated growth hormone deficiency (IGHD) in patients with an EPP. METHODS: In 67 patients (45 male) with an EPP on MRI, the site (hypothalamic vs. stalk) and surface area (SA) [ x (maximum diameter/2) x (maximum height/2), mm(2)] of the EPP were recorded and compared in patients with IGHD and MPHD in relation to clinical characteristics. RESULTS: In MPHD (n = 32) compared to IGHD (n = 35) patients: age of presentation was younger (1.4 [0.1-10.7]vs. 4.0 [0.1-11.3] years, P = 0.005), major incidents during pregnancy were increased (47%vs. 20%, P = 0.02) as were admissions to a neonatal intensive care unit (NICU) (60%vs. 26%, P = 0.04), whilst EPP SA was lower (12.3 [2.4-34.6]vs. 25.7 [6.9-48.2] mm(2), P < 0.001). In patients with a hypothalamic (n = 56) compared to a stalk sited EPP (n = 11): prevalence of MPHD was greater (55%vs. 9%,P = 0.05) and EPP surface area was smaller (17.3 [2.4-48.2]vs. 25.3 [11.8-38.5] mm(2), P < 0.001). In regression analysis, after adjusting for age, presence of MPHD was associated with: major incidents during pregnancy (RR 6.8 [95%CI 1.2-37.7]), hypothalamic EPP site (RR 10.9 [1.0-123.9]) and small EPP SA (RR 2.5 [1.0-5.0] for tertiles of SA). CONCLUSION: In patients with an EPP, adverse antenatal events, size (small) and position (hypothalamic) of the posterior pituitary gland on MRI were associated with MPHD. These findings suggest that adverse factors during pregnancy may be important for the development of an EPP.

The murine Fgfrl1 receptor is essential for the development of the metanephric kidney

Fgfrl1 is a novel member of the fibroblast growth factor receptor family. Its extracellular domain resembles the four conventional Fgfrs, while its intracellular domain lacks the tyrosine kinase domain necessary for Fgf mediated signal transduction. During embryonic development Fgfrl1 is expressed in the musculoskeletal system, in the lung, the pancreas and the metanephric kidney. Targeted disruption of the Fgfrl1 gene leads to the perinatal death of the mice due to a hypoplastic diaphragm, which is unable to inflate the lungs. Here we show that Fgfrl1-/- embryos also fail to develop the metanephric kidney. While the rest of the urogenital system, including bladder, ureter and sexual organs, develops normally, a dramatic reduction of ureteric branching morphogenesis and a lack of mesenchymal-to-epithelial transition in the nephrogenic mesenchyme result in severe renal dysgenesis. The failure of nephron induction might be explained by the absence of the tubulogenic markers Wnt4, Fgf8, Pax8 and Lim1 at E12.5 of the mutant animals. We also observed a loss of Pax2 positive nephron precursor cells and an increase of apoptosis in the cortical zone of the remnant kidney. Fgfrl1 is therefore essential for mesenchymal differentiation in the early steps of nephrogenesis.

Comparison of the receptor FGFRL1 from sea urchins and humans illustrates evolution of a zinc binding motif in the intracellular domain

BACKGROUND: FGFRL1, the gene for the fifth member of the fibroblast growth factor receptor (FGFR) family, is found in all vertebrates from fish to man and in the cephalochordate amphioxus. Since it does not occur in more distantly related invertebrates such as insects and nematodes, we have speculated that FGFRL1 might have evolved just before branching of the vertebrate lineage from the other invertebrates (Beyeler and Trueb, 2006). RESULTS: We identified the gene for FGFRL1 also in the sea urchin Strongylocentrotus purpuratus and cloned its mRNA. The deduced amino acid sequence shares 62% sequence similarity with the human protein and shows conservation of all disulfides and N-linked carbohydrate attachment sites. Similar to the human protein, the S. purpuratus protein contains a histidine-rich motif at the C-terminus, but this motif is much shorter than the human counterpart. To analyze the function of the novel motif, recombinant fusion proteins were prepared in a bacterial expression system. The human fusion protein bound to nickel and zinc affinity columns, whereas the sea urchin protein barely interacted with such columns. Direct determination of metal ions by atomic absorption revealed 2.6 mole zinc/mole protein for human FGFRL1 and 1.7 mole zinc/mole protein for sea urchin FGFRL1. CONCLUSION: The FGFRL1 gene has evolved much earlier than previously assumed. A comparison of the intracellular domain between sea urchin and human FGFRL1 provides interesting insights into the shaping of a novel zinc binding domain.

The Molecular Crosstalk between the MET Receptor Tyrosine Kinase and the DNA Damage Response - Biological and Clinical Aspects

Radiation therapy remains an imperative treatment modality for numerous malignancies. Enduring significant technical achievements both on the levels of treatment planning and radiation delivery have led to improvements in local control of tumor growth and reduction in healthy tissue toxicity. Nevertheless, resistance mechanisms, which presumably also involve activation of DNA damage response signaling pathways that eventually may account for loco-regional relapse and consequent tumor progression, still remain a critical problem. Accumulating data suggest that signaling via growth factor receptor tyrosine kinases, which are aberrantly expressed in many tumors, may interfere with the cytotoxic impact of ionizing radiation via the direct activation of the DNA damage response, leading eventually to so-called tumor radioresistance. The aim of this review is to overview the current known data that support a molecular crosstalk between the hepatocyte growth factor receptor tyrosine kinase MET and the DNA damage response. Apart of extending well established concepts over MET biology beyond its function as a growth factor receptor, these observations directly relate to the role of its aberrant activity in resistance to DNA damaging agents, such as ionizing radiation, which are routinely used in cancer therapy and advocate tumor sensitization towards DNA damaging agents in combination with MET targeting.