Characterization of the molecular mechanisms mediated by the insulin-like growth factor-2 mRNA-binding proteins and Ephrin receptor A2 (EPHA2) signaling in the malignancy of CIC-DUX4 sarcomas

Ewing sarcoma (EWS) and CIC-DUX4 sarcoma (CDS) are pediatric fusion gene-driven tumors of mesenchymal origin characterized by an extremely stable genome and limited clinical solutions. Post-transcriptional regulatory mechanisms are crucial for understanding the development of this class of tumors. RNA binding proteins (RBPs) play a crucial role in the aggressiveness of these tumors. Numerous RBP families are dysregulated in cancer, including IGF2BPs. Among these, IGF2BP3 is a negative prognostic factor in EWS because it promotes cell growth, chemoresistence, and induces the metastatic process. Based on preliminary RNA sequencing data from clinical samples of EWS vs CDS patients, three major axes that are more expressed in CDS have been identified, two of which are dissected in this PhD work. The first involves the transcription factor HMGA2, IGF2BP2-3, and IGF2; the other involves the ephrin receptor system, particularly EphA2. EphA2 is involved in numerous cellular functions during embryonic stages, and its increased expression in adult tissues is often associated with pathological conditions. In tumors, its role is controversial because it can be associated with both pro- and anti-tumoral mechanisms. In EWS, it has been shown to play a role in promoting cell migration and neoangiogenesis. Our study has confirmed that the HMGA2/IGF2BPs/IGF2 axis contributes to CDS malignancy, and Akt hyperactivation has a strong impact on migration. Using loss/gain of function models for EphA2, we confirmed that it is a substrate of Akt, and Akt hyperactivation in CDS triggers ligand-independent activation of EphA2 through phosphorylation of S897. Moreover, the combination of Trabectedin and NVP/BEZ235 partially inhibits Akt/mTOR activation, resulting in reduced tumor growth in vivo. Inhibition of EphA2 through ALWII 41_27 significantly reduces migration in vitro. The project aim is the identification of target molecules in CDS that can distinguish it from EWS and thus develop new targeted therapeutic strategies.

Correlation Between Vascular Endothelial Growth Factor Expression And Presence Of Lymph Node Metastasis In Advanced Squamous Cell Carcinoma Of The Larynx.

Squamous cell carcinoma is the most common neoplasm of the larynx, and its evolution depends on tumor staging. Vascular endothelial growth factor is a marker of angiogenesis, and its expression may be related to increased tumor aggressiveness, as evidenced by the presence of cervical lymphatic metastases. To evaluate the expression of the vascular endothelial growth factor marker in non-glottic advanced squamous cell carcinoma of the larynx (T3/T4) and correlate it with the presence of cervical lymph node metastases. Retrospective clinical study and immunohistochemical analysis of vascular endothelial growth factor through the German scale of immunoreactivity in products of non-glottic squamous cell carcinomas. This study analyzed 15 cases of advanced non-glottic laryngeal tumors (T3/T4), four of which exhibited cervical lymphatic metastases. There was no correlation between vascular endothelial growth factor expression and the presence of cervical metastases. Although vascular endothelial growth factor was expressed in a few cases, there was no correlation with the spread of cervical lymph metastases.

Vascular Endothelial Growth Factor Expression in Invasive Papillary Thyroid Carcinoma

Background: The vascular endothelial growth factor (VEGF) is a major promoter of endothelial growth and migration. Some studies have shown a correlation between expression of this growth factor and prognosis in several cancers, including well-differentiated thyroid cancer. Aim: We studied VEGF expression, local invasiveness, and other prognostic factors in papillary thyroid carcinoma (PTC) to test the hypothesis that the expression of VEGF is correlated with the degree of invasion of PTC. Patients and Methods: Clinical and pathological data of 76 patients with PTC were retrospectively reviewed. Group 1 consisted of patients with gross locally invasive tumors, group 2 consisted of patients with only invasion of the thyroid capsule, and group 3 consisted of patients with noninvasive PTC. Results: VEGF expression was noted within the tumor in all groups of PTC patients but was absent in the surrounding normal tissue. Older patients had higher expression of VEGF than younger patients. The age of patients with strong reaction to VEGF was 46 +/- 14 (mean +/- standard deviation), and that in patients with a weaker reaction was 39 +/- 16 (p<0.05). Only 20% of patients with a follicular variant of PTC had a strong reaction to VEGF compared with 68% of patients with classical PTC (p<0.01). Conclusions: VEGF expression appears to be an early event in the development of PTC. Whether VEGF expression promotes the progression of PTC is not known, but the answer to this question may be important in view of its greater expression in older patients, a group whose prognosis in PTC is worse.

In Vivo Effects on the Expression of Vascular Endothelial Growth Factor-A(165) Messenger Ribonucleic Acid of an Infrared Diode Laser Associated or Not with a Visible Red Diode Laser

Objective: This study investigated and correlated the kinetic expression of vascular endothelial growth factor (VEGF)-A(165) messenger ribonucleic acid (mRNA) with the associated use or not of an infrared laser and a visible red laser during the wound healing in rats. Background Data: There is a lack of scientific evidence demonstrating the influence of low-level laser therapy (LLLT) on the expression of VEGF mRNA in vivo. Materials and Methods: Forty-five Wistar rats were randomly allocated to one of three groups: I (n = 5, nonoperated animals), II (n = 25, operated animals), and III (n = 25, animals operated and subjected to laser irradiation). A surgical wound was performed using a scalpel in the right side of the tongue of operated animals. In group III, two sessions of laser irradiation were performed, one right after the surgical procedure (infrared laser, 780 nm, 70mW, 35 J/cm(2)) and the other 48 h later (visible red laser, 660 nm, 40mW, 5J/cm(2)). Five animals each were sacrificed 1, 3, 5, and 7 days postoperatively in groups II and III, and samples of tongue tissue were obtained. The animals of group I were sacrificed on day 7. Total RNA was extracted using guanidine-isothiocyanate-phenol-chloroform method. The results of horizontal electrophoresis after reverse transcription polymerase chain reaction permitted the ratio of VEGF-A(165) mRNA and glyceraldehyde 3-phosphate dehydrogenase mRNA expression for groups I, II, and III to be assessed (two-way analysis of variance and Tukey test, p<0.05). Results: The expression of VEGF-A(165) mRNA in group II (0.770 +/- 0.098) was statistically greater than that observed in groups I (0.523 +/- 0.164) and III (0.504 +/- 0.069) in the first day after surgery (p<0.05). Significant differences between the groups were not observed in other time periods. Conclusion: LLLT influenced the expression of VEGF-A(165) mRNA during wound healing after a surgical procedure on the tongue of Wistar rats.

Vitamin D receptor expression in the embryonic rat brain

We are interested in determining whether low maternal vitamin D-3 affects brain development in utero. Whilst the vitamin D receptor (VDR) has been identified in embryonic rat brains, the timing and magnitude of its expression across the brain remains unclear. In this study we have quantitated VDR expression during development as well correlated the timing of its appearance with two vital developmental events, apoptosis and mitosis. Brains from embryonic rats (embryonic days 15-23) were examined. We show that the well-described increase in apoptotic cells and decrease in mitotic cells during development correlates with the appearance of the VDR in brain tissue. Given that vitamin D-3 regulates mitosis and apoptosis in non-neuronal tissue we speculate that the timing of VDR expression in embryonic brain may directly or indirectly mediate features of neuronal apoptosis and mitosis.

Regulation of CSF-1 receptor expression

Cells of the mononuclear phagocyte lineage possess receptors for macrophage colony-stimulating factor (CSF-1) encoded by the c-fms protooncogene and respond to CSF-1 with increased survival, growth, differentiation, and reversible changes in function. The c-fms gene is itself a macrophage differentiation marker. In whole mount analyses of mRNA expression in embryos, c-fms is expressed at very high levels on placental trophoblasts. It is detectable on individual cells in the yolk sac around 8.5 to 9 days postcoitus, appears on isolated cells in the head of the embryo around 9.5 dpc, and appears on numerous cells throughout the embryo by day 10.5. The extent of c-fms expression is much greater than for other macrophage-specific genes including lysozyme and a macrophage-specific protein tyrosine phosphatase. Our studies of the cis-acting elements of the c-fms promoter have indicated a key role for collaboration between the macrophage-specific transcription factor, Pu.1, which functions in determining the site of transcription initiation, and other members of the Ets transcription factor family. This is emerging as a common pattern in macrophage-specific promoters. We have shown that two PU box elements alone can function as a macrophage-specific promoter. The activity of both the artifical promoter and the c-fms promoter is activated synergistically by coexpression of Pu.1 and another Ets factor, c-Ets-2. A 3.5kb c-fms exon 2 promoter (but not the 300bp proximal promoter) is also active in a wide diversity of tumor cell lines. The interesting exception is the melanoma cell line K1735, in which the promoter is completely shut down and expression of c-fms causes growth arrest and cell death. The activity of the exon 2 promoter in these nonmacrophages is at least as serum responsive as the classic serum-responsive promoter of the c-fos gene. It is further inducible in nonmacrophages by coexpression of the c-fms product. Unlike other CSF-1/c-fms-responsive promoters, the c-fms promoter is not responsive to activated Ras even when c-Ets-2 is coexpressed. In most lines, production of full length c-fms is prevented by a downstream intronic terminator, but in Lewis lung carcinoma, read-through does occur, and expression of both c-fms and other macrophage-specific genes such as lysozyme and urokinase becomes detectable in conditions of serum deprivation. (C) 1997 Wiley-Liss, Inc.

Adrenalectomy counteracts the local modulation of astroglial fibroblast growth factor system without interfering with the pattern of 6-OHDA-induced dopamine degeneration in regions of the ventral midbrain

The present study investigated the effects of bilateral adrenalectomy (ADX) on the synthesis of basic fibroblast growth factor (bFGF, FGF-2) mRNA and on the expression of its FGF receptor subtype-2 (FGFR2) mRNA after a 6-hydroxydopamine (6-OHDA)-induced lesion of nigrostriatal dopamine system. In previous papers we have demonstrated that corticosterone increases FGF-2 immunoreactivity mainly in the astrocytes of the substantia nigra [Chadi, G., Rosen, L., Cintra, A., Tinner, B., Zoli, M., Pettersson, R.F., Fuxe, K., 1993b. Corticosterone increases FGF-2 (bFGF) immunoreactivity in the substantia nigra of the rat. Neuroreport 4, 783-786.] and that 6-OHDA injected in the ventral midbrain upregulates FGF-2 synthesis in reactive astrocytes in the ascending dopamine pathways [Chadi, G., Cao, Y., Pettersson, R.F., Fuxe, K., 1994. Temporal and spatial increase of astroglial basic fibroblast growth factor synthesis after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine neurons. Neuroscience 61, 891-910.]. Rats were adrenalectomized and received a 6-OHDA stereotaxical injection in the ventral midbrain 2 days later. Seven days after the dopamine lesion, Western blot analysis showed a decreased level of tyrosine hydroxylase in the lesioned side of the midbrain, an event that was not altered by ADX or corticosterone replacement. Moreover, the degeneration of nigral dopamine neurons, which was confirmed by the disappearance of acidic FGF (FGF-1) mRNA and the decrement of tyrosine hydroxylase mRNA labeled nigral neurons, was not altered by ADX. The FGF-2 protein (23 kDa isoform but not 21 kDa fraction) levels increased in the lesioned side of the ventral midbrain. This elevation was counteracted by ADX, an effect that was fully reversed by corticosterone replacement. In situ hybridization revealed that ADX counteracted the elevated FGF-2 mRNA levels in putative glial cells of the ipsilateral pars compacta of the substantia nigra and in the ventral tegmental area. The ADX also counteracted the increased density and intensity of the astroglial FGF-2 immunoreactive profiles within the lesioned pars compacta of the substantia nigra and the ventral tegmental area as determined by stereology. The stereotaxical mechanical needle insertion triggered the expression of FGFR2 mRNA in putative glial cells, spreading to the entire ipsilateral ventral midbrain from the region of needle track, an occurrence that was partially reversed by ADX. In conclusion, bilateral ADX counteracted the increased astroglial FGF-2 synthesis in the dopamine regions of the ventral midbrain following a 6-OHDA-induced local lesion and interfered with FGF receptor regulation around injury. These findings give further evidence that adrenocortical hormones may regulate the astroglial FGF-2-mediated trophic mechanisms and wound repair events in the lesioned central nervous system. (c) 2007 Elsevier B.V. All rights reserved.

Assessment of the Expression of IR beta, IRS-1, IRS-2 and IGF-IR beta in a Rat Model of Intrauterine Growth Restriction

Objective: To investigate glomerular development and expression of insulin and insulin-like growth factor receptors in an experimental model of intrauterine growth restriction (IUGR). Material and Methods: We studied three groups of Sprague-Dawley fetuses: IUGR - restricted by ligation of the right uterine artery; C-IUGR - left horn controls, and EC - external controls (non-manipulated). Body and organs were weighed, and glomerular number and volume were analyzed. Expression of IR beta, IRS-1, IRS-2 and IGF-IR beta was analyzed in liver, intestine and kidneys by immunoblotting. Results: Organ/body weight ratios were similar. In IUGR, glomerular number and volume were increased compared to C-IUGR and EC (p < 0.001). In the IUGR liver, increases were found in IGF-IR beta compared to C-IUGR and EC; IR beta compared to EC, and IRS-2 compared to C-IUGR. However, decreases in IR beta were noted in IUGR compared to C-IUGR; IRS-1 compared to C-IUGR and EC, and IRS-2 compared to EC. In IUGR intestine, increases were detected in IR beta, IRS-1 and IGF-IR beta compared to C-IUGR and EC. In IUGR kidneys, increases were observed in IR beta and IGF-IR beta compared to C-IUGR and EC, and IRS-1 compared to EC. Decreased IRS-2 in the intestine and kidney were noticed in IUGR compared to C-IUGR and EC. Conclusion: IUGR fetuses had less glomeruli and alterations in insulin receptors, which may be associated with an increased risk of disease occurrence in adulthood. Copyright (C) 2010 S. Karger AG, Basel

Expression of fibroblast growth factor receptors during development and regression of the bovine corpus luteum

There is evidence that fibroblast growth factors (FGFs) are involved in the regulation of growth and regression of the corpus luteum (CL). However, the expression pattern of most FGF receptors (FGFRs) during CL lifespan is still unknown. The objective of the present study was to determine the pattern of expression of `B` and `C` splice variants of FGFRs in the bovine CL. Bovine CL were collected from an abattoir and classed as corpora hemorrhagica (Stage I), developing (Stage II), developed (Stage III) or regressed (Stage IV) CL. Expression of FGFR mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction and FGFR protein was localised by immunohistochemistry. Expression of mRNA encoding the `B` and `C` spliced forms of FGFR1 and FGFR2 was readily detectable in the bovine CL and was accompanied by protein localisation. FGFR1C and FGFR2C mRNA expression did not vary throughout CL lifespan, whereas FGFR1B was upregulated in the developed (Stage III) CL. FGFR3B, FGFR3C and FGFR4 expression was inconsistent in the bovine CL. The present data indicate that FGFR1 and FGFR2 splice variants are the main receptors for FGF action in the bovine CL.

Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: Immunolocalization according to the odontogenesis phases

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has several biological effects in vivo including control of cell growth and differentiation, cell migration, lineage determination, motility, adhesion, apoptosis, and synthesis and degradation of extracellular matrix, and TGF-beta plays an important role in regulating tissue repair and regeneration. Our study analyzed the participation of TGF-beta 1, -beta 2, and -beta 3 in the different stages of morphogenesis and differentiation of human developing dental organ using immunobistochemistry. The maxillae and mandibles of 10 human embryos ranging from 8 to 23 weeks of gestation were employed, according to the approval of the ethical committee. Our study revealed that the TGF-beta subunits-beta 1, beta 2, and beta 3 were present in the various stages of tooth development, but the expression varied according to the differentiation stage, tissue, and TGF-beta subunit. Our results indicated that TGF-beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF-beta 2 to cellular differentiation, and TGF-beta 3 to mineral maturation matrix.

Insulin-like growth factor 2 cDNA cloning and ontogeny of gene expression in the liver of the marsupial brushtail possum (Trichosurus vulpecula)

The cDNA sequence for insulin-like growth factor 2 (IGF-2) was determined from the liver of the marsupial brushtail possum (Trichosurus vulpecula) using reverse transcription followed by polymerase chain reaction (RT-PCR) with gene-specific primers. The 359 bp of possum sequence encompassed the mature peptide, 27 bp of the signal peptide, and 125 bp of the E-peptide. Alignment of the deduced amino acid sequence with those from other species indicated that the mature peptide was 71 amino acids in length, 4 amino acids longer than most other mammals. At both the nucleotide and amino acid levels there was a high degree of sequence identity with IGF-2 from other mammalian and nonmammalian species. Amino acid identity ranged from 94.4% with a variant form of human IGF-2 to 80.3% with zebrafinch IGF-2. Northern analysis revealed that radiolabeled possum IGF-2, cDNA hybridized to multiple transcripts in the liver of both adult possums and 150-day-old pouch young and that the overall level of expression was greater in pouch young. Semiquantitative RT-PCR with total RNA from liver samples of pouch young aged 12 to 150 days postpartum and adults confirmed that IGF-2 gene expression was two to three times more abundant in pouch young than in adults but there was no significant change in the level of expression during pouch life. Unlike other mammalian species, in which there is a decline in levels of liver IGF-2 gene expression around the time of birth, levels in the marsupial brushtail possum remain elevated for at least 150 days after birth. This suggests that the decline in liver IGF-2 expression in marsupials and eutherians occurs at a similar stage of development and may reflect a role for this growth factor during the postnatal growth and development of the marsupial, (C) 2001 Academic Press.

Growth hormone (GH)/GH receptor expression and GH-mediated effects during early bovine embryogenesis

Pituitary growth hormone (GH) stimulates postnatal growth and metabolism. The role of CH and its receptor (GHR) during prenatal development, however, is still controversial. As shown by reverse transcription polymerase chain reaction (RT-PCR), bovine in vitro fertilization embryos synthesized the transcript of GHR from Day 2 of embryonic life onwards. Real time RT-PCR revealed that synthesis of GHR mRNA was increased 5.9-fold in 6-day-old embryos compared with 2-day-old embryos. Using in situ hybridization, the mRNA encoding GHR was predominantly localized to the inner cell mass of blastocysts. The GHR protein was first visualized 3 days after fertilization. GH-specific transcripts were first detected in embryos on Day 8 of in vitro culture. As shown by transmission electron microscopy, GH treatment resulted in elimination of glycogen storage in 6- to 8-day-old embryos and an increase in exocytosis of lipid vesicles. These results suggest that a functional GHR able to modulate carbohydrate and lipid metabolism is synthesized during preimplantation development of the bovine embryo and that this GHR may be subject to activation by embryonic GH after Day 8.

The expression of fibroblast growth factors and their receptors in the embryonic and neonatal mouse inner ear

Four different fibroblast growth factor receptors (FGFR) are known, three of which have splice variants (known as the b and c variants) in the FGF-binding domain, to give different patterns of sensitivity to the different FGFs. The expression of the b and c variants of the FGF receptors. together with the expression of the ligands FGF1. FGF2, FGF3, FGF7, FGF8b and FGF8c, was determined by quantitative reverse transcription-polymerase chain reaction in developing whole mouse inner ears, and in dissected components of the postnatal mouse inner ear. At embryonic age (E)10.5 days, when the otocyst is a simple closed sac, the receptor most heavily expressed was FGFR2b, relative to the postnatal day 0 level. Over the period E10.5-E12.5. during which the structures of the inner ear start to form, the expression of the different FGF receptors increased 10(2)-10(4) fold per unit of tissue, and there was a gradual switch towards expression of the 'c' splice variants of FGFR2 and FGFR3 rather than the 'b' variants. At E10.5, the ligands most heavily expressed, relative to the postnatal day 0 level, were FGF3, FGF8b and FGF8c. In the postnatal inner eat. the patterns of expression of receptors and ligands tended to be correlated, such that receptor variants were expressed in the same regions as the ligands that are known to activate them effectively. The neural/sensory region expressed high levels of FGFR3c, and high levels of the ligand FGF8b. The same area also expressed high levels of FGFR1b and FGFR2b, and high levels of FGF3. The lateral wall of the cochlea (including the stria vascularis and the spiral ligament) expressed high levels of FGFR1c and FGF1. 11 is suggested that the different FGF receptors and ligands are expressed in a spatially coordinated pattern to selectively program cochlear development. (C) 2001 Elsevier Science B.V. All rights reserved.

Growth hormone receptor and IGF-1 receptor immunoreactivity during orthodontic tooth movement in the prednisolone-treated rat

Bone remodeling during tooth movement is regulated by local and systemic factors. Two regulators of bone metabolism are growth hormone (GH) and insulin-like growth factor-I (IGF-1). Their effects are mediated via binding to GH receptor (GHR) and IGF-I receptor (IGF-IR) in target tissues. Corticosteroids may affect the activity of these growth factors. This study examined the effect of prednisolone on GHR and IGF-IR expression in dental tissues following orthodontic tooth movement. The corti ticosteroid-treated group (N = 6) was administered prednisolone ( 1 mg/kg,) daily and the control group (N = 6) received equivalent volumes of saline. An orthodontic force (30 g) was applied to the maxillary first molar. Animals were sacrificed 12 days postappliance insertion. Sagittal sections of the first molar were stained for GHR and IGF-IR immunoreactivity. GHR and IGF-IR cell counts were elevated following appliance-treatment. Orthodontic tooth movement appeared to up-regulate GHR and IGF-IR immunoreactivity, but this up-regulation was reduced following prednisolone treatment. The suppression of GHR and IGF-I immunoreactivity in steroid-treated animals infers the mechanism whereby bone resorption and deposition, necessary for orthodontic tooth movement, may be inhibited by prednisolone. However, at 12 days postappliance insertion. no difference in orthodontic tooth movement was observed following low-dose prednisolone treatment.