Christine Delfino

Effects of adrenomedullin on endothelial cells in the multistep process of angiogenesis: Involvement of CRLR/RAMP2 and CRLR/RAMP3 receptors

Recently, we demonstrated that U87 glioblastoma xenograft tumors treated with anti‐adrenomedullin (AM) antibody were less vascularized than control tumors, suggesting that AM might be involved in neovascularization and/or vessel stabilization. Angiogenesis, the sprouting of new capillaries from preexisting blood vessels, is a multistep process that involves migration and proliferation of endothelial cells, remodeling of the extracellular matrix and functional maturation of the newly assembled vessels. In our study, we analyzed the role of AM on human umbilical vein endothelial cell (HUVEC) phenotype related to different stages of angiogenesis. Here we report evidence that AM promoted HUVEC migration and invasion in a dose‐dependent manner. The action of AM is specific and is mediated by the calcitonin receptor‐like receptor/receptor activity‐modifying protein‐2 and ‐3 (CRLR/RAMP2; CRLR/RAMP3) receptors. Furthermore, AM was able to induce HUVEC differentiation into cord‐like structures on Matrigel. Suboptimal concentrations of vascular endothelial growth factor (VEGF) and AM acted synergistically to induce angiogenic‐related effects on endothelial cells in vitro. Blocking antibodies to VEGF did not significantly inhibit AM‐induced capillary tube formation by human endothelial cells, indicating that AM does not function indirectly through upregulation of VEGF. These findings suggest that the proangiogenic action of AM on cultured endothelial cells via CRLR/RAMP2 and CRLR/RAMP3 receptors may translate in vivo into enhanced neovascularization and therefore identify AM and its receptors acting as potential new targets for antiangiogenic therapies.

Targeting adrenomedullin receptors with systemic delivery of neutralizing antibodies inhibits tumor angiogenesis and suppresses growth of human tumor xenografts in mice

Adrenomedullin (AM) is a multifunctional peptide vasodilator that transduces its effects through calcitonin receptor‐like receptor/receptor activity modifying protein‐2 and ‐3 (CLR/RAMP2 and CLR/ RAMP3). Previously, we reported on the development of an anti‐AM antibody that potently inhibits tumor cell proliferation in vitro and tumor growth in vivo. Here, we report the effect of anti‐AM receptor antibodies (αAMRs) on angiogenesis and tumor growth. We demonstrate that αAMRs decrease in a dose‐dependent manner the growth of U87 glioblastoma cells and HT‐29 colorectal cancer cells, but not A549 lung cancer cells, in vitro. In vivo, AM in Matrigel plugs induces angiogenesis by promoting recruitment of endothelial cells, pericytes, myeloid precursor cells, and macrophages and by promoting channel formation. Remarkably, systemic administration of αAMRs every 3 d markedly reduced neovascularization of Matrigel plugs in a dosedependent fashion, as demonstrated by reduced numbers of the recruited cells and vessel structures. Several human tumor xenografts in athymic mice were used to examine the effect of αAMR treatment on tumor angiogenesis and growth. αAMR treatment significantly suppressed the growth of glioblastoma, lung, and colon tumors. Histological examination of αAMR‐treated tumors showed evidence of disruption of tumor vascularity with decreased microvessel density, depletion of endothelial and pericyte cells, and increased tumor cell apoptosis. These findings support the conclusion that αAMR treatment inhibits tumor growth by suppression of angiogenesis and tumor growth and suggest that AMRs may be useful therapeutic targets.—Kaafarani, I., Fernandez‐Sauze, S., Berenguer, C., Chinot, O., Delfino, C., Dussert, C., Metellus, P., Boudouresque, F., Mabrouk, K., Grisoli, F., Figarella‐Branger, D., Martin, P.‐M., Ouafik, L. H. Targeting adrenomedullin receptors with systemic delivery of neutralizing antibodies inhibits tumor angiogenesis and suppresses growth of human tumor xenografts in mice. FASEB J. 23, 3424–3435 (2009). www.fasebj.org

Expression and role of adrenomedullin in renal tumors and value of its mRNA levels as prognostic factor in clear-cell renal carcinoma

Antiangiogenic therapies are used for advanced clear‐cell renal carcinomas (cRCC), but without curative possibilities, underlining the need for new therapeutic targets. Adrenomedullin (AM), a multifunctional peptide, is highly expressed in several tumors and plays an important role in angiogenesis and tumor growth through its receptors: calcitonin receptor‐like receptor/receptor activity‐modifying protein 2 and 3 (CLR/RAMP2 and CLR/RAMP3). In this study, real‐time quantitative reverse‐transcription‐PCR showed AM mRNA levels were higher in cRCC and in chromophobe renal carcinomas (chRCC) than in normal renal tissue. Interestingly, AM mRNA expression in cRCC correlated strongly with VEGF‐A mRNA expression. Immunohistochemically, AM, CLR and RAMP2 were localized in the carcinomatous epithelial compartment of cRCC. Interestingly, RAMP3 immunostaining was found only in the inflammatory cells that infiltrated tumors, suggesting a cross talk between tumor cells and the microenvironment. We also observed that cRCC cells BIZ and 786‐O expressed and secreted AM into the culture medium. In vitro, exogenous AM treatment stimulated cell proliferation, migration and invasion, indicating the cell can respond to AM. The action of AM was specific and was mediated by the CLR/RAMP2 and CLR/RAMP3 receptors. Clinical data showed the prognostic value of AM. High AM mRNA levels were associated with an increased risk of relapse after curative nephrectomy for cRCC. These findings highlight the implication of the AM pathway in the metastatic process and the prognostic relevance of AM in cRCC and point to a potential new therapeutic target.

Evaluation of quantitative measurement of thyroglobulin mRNA in the follow-up of differentiated thyroid cancer.

Detection of thyroid cancer by thyroglobulin (Tg) assay in peripheral blood is useful in the absence of residual thyroid tissue, but it requires thyrotropin stimulation for maximal sensitivity and is affected by circulating antithyroglobulin antibodies. To avoid these drawbacks, thyroglobulin mRNA (Tg mRNA) assay in circulating blood has been proposed. Initial studies showed that Tg mRNA assay was more positive in patients with metastasis than in cured patients. Further studies showed controversial data. We measured Tg mRNA in 26 patients undergoing levothyroxine (LT(4)) suppressive therapy after total thyroidectomy for thyroid cancer and in 11 controls. The stage of the cancer was defined according to the findings of the latest whole-body (131)I scan and serum Tg performed under LT(4) withdrawal. Patients were classified as cured (negative scan, negative stimulated Tg, 8 patients), with metastasis (positive scan in extrathyroid bed regions, positive Tg, 7 patients), with thyroid remnants (positive scan in thyroid bed, positive Tg, 8 patients), and discordant cases (negative scan, positive Tg, 3 patients). RNA was extracted from blood and analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) using two sets of primers and internal probes specific for Tg mRNA. This method allowed the detection of Tg mRNA in thyroid biopsies. Tg mRNA was undetectable in all control subjects and in all patients with cured cancer, positive in 1 of 8 patients with thyroid remnants, and in only 1 of 7 patients with metastasis. In conclusion, our data do not support the usefulness of Tg mRNA measurements in blood for monitoring thyroid cancer.

Mammalian peptidylglycine alpha-amidating monooxygenase mRNA expression can be modulated by the La autoantigen.

ABSTRACT Peptidylglycine α-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal α-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3′ untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3′ UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3′ UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3′ UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules.

Estrogen Regulation of Peptidylglycine α-Amidating Monooxygenase Expression in Anterior Pituitary Gland

The pituitary is a rich source of peptidylglycine α-amidating monooxygenase (PAM). This bifunctional protein contains peptidylglycineα -hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxyglycineα -amidating lyase catalytic domains necessary for the two-step formation of α-amidated peptides from their COOH-terminal glycine extended precursors. Expression of PAM was evaluated in the anterior pituitary of intact cycling adult female rat and after experimental manipulation of estrogen status. PAM messenger RNA (mRNA) levels showed changes inversely related to the physiological variations of plasma estrogen levels during the estrous cycle. Chronic treatment of ovariectomized (OVX) rats with 17 β-estradiol decreased PAM mRNA levels to values comparable with those found in intact rats at proestrus. In situ hybridization of anterior pituitary sections using 35S-labeled full length RNA antisense transcripts of rat PAM-1 complementary DNA showed that 17 β-estradiol treatment induced an overall decrease of the ...

Expression of adrenomedullin in human colorectal tumors and its role in cell growth and invasion in vitro and in xenograft growth in vivo

Adrenomedullin (AM) is a multifunctional peptide vasodilator that transduces its effects through calcitonin receptor‐like receptor/receptor activity‐modifying protein‐2 and ‐3 (CLR/RAMP2 and CLR/RAMP3). In this study, real‐time quantitative reverse transcription demonstrated a significant expression of AM mRNA in tumor samples from colorectal cancer (CRC) patients in clinical stage II, III, and IV when compared with normal colorectal tissue. AM, CLR, RAMP2, and RAMP3 proteins were immunohistochemically localized in the carcinomatous epithelial compartment of CRC tissue. Tissue microarray analysis revealed a clear increase of AM, CLR, RAMP2, and RAMP3 staining in lymph node and distant metastasis when compared with primary tumors. The human colon carcinoma cells HT‐29 expressed and secreted AM into the culture medium with a significant increase under hypoxia. Treatment of HT‐29 cells with synthetic AM stimulated cell proliferation and invasion in vitro. Incubation with anti‐AM antibody (αAM), anti‐AM receptors antibodies (αAMR), or AM antagonist AM22–52 inhibited significantly basal levels of proliferation of HT‐29 cells, suggesting that AM may function as an autocrine growth factor for CRC cells. Treatment with αAM significantly suppressed the growth of HT‐29 tumor xenografts in vivo. Histological examination of αAM‐treated tumors showed evidence of disruption of tumor vascularity with decreased microvessel density, depletion of endothelial cells and pericytes, and increased tumor cell apoptosis. These findings highlight the potential importance of AM and its receptors in the progression of CRC and support the conclusion that αAM treatment inhibits tumor growth by suppression of angiogenesis and tumor growth, suggesting that AM may be a useful therapeutic target.

Identification of secondary structure in the 5′-untranslated region of the human adrenomedullin mRNA with implications for the regulation of mRNA translation

Adrenomedullin (AM) is a multifunctional regulatory peptide with important angiogenic and mitogenic properties. Here we identify a region of stable secondary structure in the 5′-untranslated region (5′ UTR) of human AM mRNA. Reverse transcriptase–polymerase chain reaction of the 5′ UTR consistently resulted, in addition to the product with the expected size of 155 base pair (bp), in a second product with an ∼65-bp deletion from the central region of the 5′ UTR, suggesting the presence of a secondary structure. The presence of a stem–loop structure was confirmed by probing the 5′ UTR with RNases with selectivity for single- or double-stranded RNA. We investigated the role of this stem–loop structure in expression of luciferase reporter gene in cultured cell lines. Reporter assays using a chimeric mRNA that combined luciferase and the 5′ UTR of AM mRNA demonstrated a dramatic decrease of the reporter activity owing to a decreased translation, whereas the deletion of the stem–loop structure localized between nt +31 and +95 from the cap site led to the recovery of activity. Gel migration shift assays using cytosolic extracts from mammalian cell lines demonstrate a specific binding of a cytosolic protein to riboprobes containing the 5′ UTR of AM but not to riboprobes either corresponding to other areas of the message or containing the 5′ UTR but lacking the region of secondary structure. Although we conclude that the 5′ UTR of the human AM mRNA can modulate the translation of AM mRNA in vivo, and that the predicted stem–loop structure is necessary for this inhibition, the functional consequences of the cis element-binding activity remain to be determined.

Stromal fibroblasts present in breast carcinomas promote tumor growth and angiogenesis through adrenomedullin secretion

Tumor- or cancer-associated fibroblasts (TAFs or CAFs) are active players in tumorigenesis and exhibit distinct angiogenic and tumorigenic properties. Adrenomedullin (AM), a multifunctional peptide plays an important role in angiogenesis and tumor growth through its receptors calcitonin receptor-like receptor/receptor activity modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). We show that AM and AM receptors mRNAs are highly expressed in CAFs prepared from invasive breast carcinoma when compared to normal fibroblasts. Immunostaining demonstrates the presence of immunoreactive AM and AM receptors in the CAFs (n = 9). The proliferation of CAFs is decreased by anti-AM antibody (αAM) and anti-AM receptors antibody (αAMR) treatment, suggesting that AM may function as a potent autocrine/paracrine growth factor. Systemic administration of αAMR reduced neovascularization of in vivo Matrigel plugs containing CAFs as demonstrated by reduced numbers of the vessel structures, suggesting that AM is one of the CAFs-derived factors responsible for endothelial cell-like and pericytes recruitment to built a neovascularization. We show that MCF-7 admixed with CAFs generated tumors of greater volume significantly different from the MCF-7 xenografts in nude mice due in part to the induced angiogenesis. αAMR and AM22-52 therapies significantly suppressed the growth of CAFs/MCF-7 tumors. Histological examination of tumors treated with AM22-52 and aAMR showed evidence of disruption of tumor vasculature with depletion of vascular endothelial cells, induced apoptosis and decrease of tumor cell proliferation. Our findings highlight the importance of CAFs-derived AM pathway in growth of breast carcinoma and in neovascularization by supplying and amplifying signals that are essential for pathologic angiogenesis.

Molecular profile of androgen-independent prostate cancer xenograft LuCaP 23.1

After castration or therapeutic hormone deprivation, most cancer of the prostate (CaP) cells develop androgen-independent (AI) growth. In this work, we studied the effect of androgen depletion (castration) on the growth of experimental model LuCaP 23.1 xenograft. A total of 101 nude mice were implanted and analysed for their growth profile before experimental period 1 (11 weeks) and after castration experimental period 2 (15 weeks). For specific periods, tumors were harvested and assessed for molecular marker expression specific for CaP. Taking into account tumor dynamic growth, prior to castration we found 37 fast growing (FG) tumors (948.9+/-76.9 mm3) and 63 slow growing (SG) tumors (229.6+/-18.4 mm3). Real-time quantitative RT-PCR showed that in comparison to SGs, FGs contained elevated expression of epidermal growth factor receptor type 1 (HER1), urokinase plasminogen activator (uPA), thymidine phosphorylase (TP) and thymidilate synthase (TS) mRNAs expression and low levels of 5alpha-reductase 2 (5alpha-R2) mRNA. After castration all FG tumors progressed rapidly (by 5 weeks) to AI growth (FG-P). In SG castrated tumors, 66% of tumors showed retarded progression (by 12 weeks) to AI (SG-P), whereas 34% responded to castration (SG-R). Molecular analysis demonstrated distinct molecular profiles integrating different pathways associated with AI progression. The progressive tumors FG-P, and some tumors of SG-P subgroup, presented significantly high levels of HER1, epidermal growth factor receptor type 2 (HER2), TS, uPA, TP, tumor necrosis factor superfamily member 6 (FAS) and peptidylglycine alpha-amidating mono-oxygenase (PAM) mRNA all of which correlated with androgen receptor (AR) mRNA. The second subgroup of SG-P tumors showed a high expression of the anti-apoptotic gene Bcl-2. A third subgroup of SG-P tumors showed significant expression of hypoxia-related genes such as adrenomedullin (AM) after castration. LuCaP 23.1 xenograft represent a useful dynamic model to study pre-clinically new therapeutic molecules and evaluate non-randomized therapeutics protocols combining different target inhibition specific to each AI pathways.