Clara Nahmias

Angiotensin receptors: a new role in cancer?

There is increasing evidence that Angiotensin II (AngII), a major regulator of blood pressure and cardiovascular homeostasis, is involved in the regulation of cell proliferation, angiogenesis, inflammation and tissue remodeling, which suggests that this peptide might also play a role in cancer. This review focuses on the expression and function of Angiotensin I-converting enzyme (ACE) and AngII receptors in various aspects of cancer. Recent experimental data suggests that ACE inhibitors and AngII type 1 receptor (AT1R) antagonists have beneficial effects on tumor progression, vascularization and metastasis, and that the AngII type 2 receptor (AT2R) subtype has a potential role in cancer. An overview of the major intracellular signaling pathways associated with AT1R and AT2R activation in cancer cells, as well as in endothelial and inflammatory cells, is presented here.

Molecular characterization of the mouse beta 3-adrenergic receptor: relationship with the atypical receptor of adipocytes.

The gene encoding the murine beta 3‐adrenergic receptor (beta 3AR) has been isolated. It translates into a polypeptide of 388 amino acid residues which shows 82% overall homology with the human beta 3AR. In Southern blot experiments, a probe derived from the murine beta 3AR gene hybridizes to a unique restriction fragment in the murine and human genomes. In both species, the beta 3AR gene is located on chromosome 8, in regions (8A2‐‐‐‐8A4 in mouse, and 8p11‐‐‐‐8p12 in man) which are conserved between mouse and man. The pharmacological profile of the mouse beta 3AR strongly resembles that of the human beta 3AR. It is characterized by a low affinity toward the radiolabelled beta‐adrenergic antagonist [125I]Iodocyanopindolol and a low efficiency of other antagonists such as propranolol, ICI 118551 or CGP 20712A to inhibit cAMP production induced by isoproterenol. Another salient feature shared by the murine and the human beta 3ARs is the very potent effect of the lipolytic compound BRL 37344 on cAMP accumulation and the partial agonistic effect of the beta 1‐ and beta 2‐adrenergic antagonists CGP 12177A, oxprenolol and pindolol. These properties are very close to those ascribed to the atypical beta AR of rodent adipocytes. In addition, Northern blot analyses indicate that the beta 3AR gene is mainly expressed in mouse brown and white adipose tissues, suggesting that the murine beta 3AR described here is the atypical beta AR involved in the control of energy expenditure in fat tissue.

Signal transduction from the angiotensin II AT2 receptor.

Recent studies of genetically engineered animals have established a role for the angiotensin II (AT2) receptor in cardiovascular, renal and central functions, as well as in developmental processes. This review summarizes new insights into major AT2 signaling pathways--activation of protein phosphatases, the nitric oxide-cGMP system and phospholipase A2--which have been related to specific cellular responses or functions of this receptor.

The Tyrosine Phosphatase SHP-1 Associates with the sst2 Somatostatin Receptor and Is an Essential Component of sst2-mediated Inhibitory Growth Signaling

Activation of the somatostatin receptor sst2, a member of the Gi protein-coupled receptor family, results in the stimulation of a protein-tyrosine phosphatase activity involved in the sst2-mediated growth inhibitory signal. Here, we report that SHP-1, a cytoplasmic protein-tyrosine phosphatase containing two Src homology 2 domains constitutively associated with sst2 as evidence by coprecipitation of SHP-1 protein with sst2, in Chinese hamster ovary cells coexpressing sst2 and SHP-1. Activation of sst2 by somatostatin resulted in a rapid dissociation of SHP-1 from sst2 accompanied by an increase of SHP-1 activity. SHP-1 was phosphorylated on tyrosine in control cells and somatostatin induced a rapid and transient dephosphorylation on tyrosine residues of the enzyme. Stimulation of SHP-1 activity by somatostatin was abolished by pertussis toxin pretreatment of cells. Giα3 was specifically immunoprecipitated by anti-sst2 and anti-SHP-1 antibodies, and somatostatin induced a rapid dissociation of Giα3 from sst2, suggesting that Giα3 may be involved in the sst2·SHP-1 complexes. Finally, somatostatin inhibited the proliferation of cells coexpressing sst2 and SHP-1, and this effect was suppressed in cells coexpressing sst2 and the catalytic inactive SHP-1 (C453S mutant). Our data identify SHP-1 as the tyrosine phosphatase associated with sst2 and demonstrate that this enzyme may be an initial key transducer of the antimitogenic signaling mediated by sst2.

Pivotal role of tyrosine phosphatase SHP-1 in AT2 receptor-mediated apoptosis in rat fetal vascular smooth muscle cell

OBJECTIVE To examine the possible crosstalk and the roles of angiotensin (Ang) II type 1 (AT1) and type 2 (AT2) receptors in the control of apoptosis in fetal vascular smooth muscle cells (VSMCs). METHODS Fetal VSMCs were prepared from rat fetal aorta at embryonic day 20. Expression of Ang II receptors was measured by a radioligand binding assay. Apoptotic changes were assessed by caspase 3 activity and chromatin dye staining. Regulation of extracellular signal-regulated kinase (ERK) activity via Ang II receptors was analysed by determining phosphorylated ERK with Western blot. Ang II receptor-mediated activation of tyrosine phosphatase SHP-1 was assessed by protein tyrosine phosphatase assay. RESULTS The expression of AT1 and AT2 receptors was approximately 70%: 30% per cell. Serum depletion induced apoptosis in fetal VSMCs and selective AT1 receptor stimulation attenuated the apoptotic changes, whereas selective AT2 receptor activation enhanced apoptosis. Ang II increased ERK phosphorylation, which was inhibited by addition of the AT1 receptor-specific antagonist CV11974, but enhanced by addition of the AT2 receptor-specific antagonist PD123319, suggesting that activation of AT2 receptor attenuated the AT1 receptor-mediated ERK phosphorylation. Moreover, we demonstrated that AT2 receptor stimulation activated SHP-1 in fetal VSMCs, whereas AT1 receptor stimulation did not. Transient transfection of a dominant-negative SHP-1 mutant into rat fetal VSMCs resulted in a significant decrease of the AT2 receptor-mediated inhibition of ERK phosphorylation and attenuated the proapoptotic effect of AT2 receptor. CONCLUSION These results indicate that a crosstalk between AT1 and AT2 receptors regulates the survival of fetal VSMCs and substantiate SHP-1 as a key molecule in AT2 receptor signaling.

Angiotensin II Facilitates Breast Cancer Cell Migration and Metastasis

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pre-treatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.

Angiotensin II type 2 receptor inhibits epidermal growth factor receptor transactivation by increasing association of SHP-1 tyrosine phosphatase

Angiotensin (Ang) II has 2 major receptor isoforms, Ang type 1 (AT1) and Ang type (AT2). AT1 transphosphorylates epidermal growth factor receptor (EGFR) to activate extracellular signal–regulated kinase (ERK). Although AT2 was shown to inactivate ERK, the action of AT2 on EGFR activation remains undefined. Using AT2-overexpressing vascular smooth muscle cells from AT2 transgenic mice, we studied these undefined actions of AT2. Maximal ERK activity induced by Ang II was increased 1.9- and 2.2-fold by AT2 inhibition, which was abolished by orthovanadate but not okadaic acid or pertussis toxin. AT2 inhibited AT1-mediated EGFR tyrosine phosphorylation by 63%. The activity of SHP-1 tyrosine phosphatase was significantly upregulated 1 minute after AT2 stimulation and association of SHP-1 with EGFR was increased, whereas AT2 failed to tyrosine phosphorylate SHP-1. Stable overexpression of SHP-1–dominant negative mutant completely abolished AT2-mediated inhibition of EGFR and ERK activation. AT1-mediated c-fos mRNA accumulation was attenuated by 48% by AT2 stimulation. Induction of fibronectin gene containing an AP-1 responsive element in its 5′-flanking region was decreased by 37% after AT2 stimulation, corresponding to the results of gel mobility assay with the AP-1 sequence of fibronectin as a probe. These findings suggested that AT2 inhibits ERK activity by inducing SHP-1 activity, leading to decreases in AP-1 activity and AP-1–regulated gene expression, in which EGFR dephosphorylation plays an important role via association of SHP-1.

An ATIPical family of angiotensin II AT2 receptor-interacting proteins

AT2, the second subtype of angiotensin II receptors, is a major component of the renin-angiotensin system involved in cardiovascular and neuronal functions. AT2 belongs to the superfamily of G protein-coupled receptors, but its intracellular signaling pathways have long remained elusive. Over the past few years, efforts to characterize this atypical receptor have led to the identification of novel molecular scaffolds that directly bind to its intracellular tail. The present review focuses on a family of AT2 receptor-interacting proteins (ATIPs) involved in neuronal differentiation, vascular remodeling and tumor suppression. Recent findings that ATIPs and ATIP-related proteins associate with microtubules suggest that they might constitute a novel family of multifunctional proteins regulating a wide range of physiopathological functions.

The metastatic microenvironment: brain-residing melanoma metastasis and dormant micrometastasis.

Brain metastasis occurs frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. We generated a reproducible melanoma brain metastasis model, consisting of brain‐metastasizing variants and local, subdermal variants that originate from the same melanomas thus sharing a common genetic background. The brain‐metastasizing variants were obtained by intracardiac inoculation. Brain metastasis variants when inoculated subdermally yielded spontaneous brain dormant micrometastasis. Cultured cells from the spontaneous brain micrometastasis grew very well in vitro and generated subdermal tumors after an orthotopic inoculation. Expression analysis assays indicated that the brain metastasis and micrometastasis cells expressed higher levels of angiopoietin‐like 4, prostaglandin‐synthesizing enzyme cyclooxygenase‐2, matrix metalloproteinase‐1 and preferentially expressed antigen in melanoma and lower levels of claudin‐1 and cysteine‐rich protein 61 than the corresponding cutaneous variants. The reproducible models of human melanoma metastasizing experimentally and spontaneously to the brain will facilitate the identification of novel biomarkers and targets for therapy and contribute to the deciphering of mechanisms underlying melanoma metastasis.

Mutation analysis of the 8p22 candidate tumor suppressor gene ATIP/MTUS1 in hepatocellular carcinoma.

A high frequency of allelic loss affecting chromosome 8p and a minimal region of deletion at p21-22 have been previously reported in hepatocellular carcinoma (HCC), suggesting that at least one tumor suppressor gene is present in this region. In this study, we assessed whether the angiotensin II AT2 receptor interacting protein (ATIP)/mitochondrial tumor suppressor gene (MTUS1), a gene newly identified at position 8p22, may be a candidate tumor suppressor gene mutated in HCC. We searched for alterations in the 17 coding exons of ATIP/MTUS1 by means of denaturating high-performance liquid chromatography and sequencing, in 51 HCC tumors and 58 cell lines for which loss of heterozygosity status was known. Five major nucleotide substitutions were identified, all located in exons used by the ATIP3 transcript which is the only ATIP transcript variant expressed in liver. These nucleotide variations result in amino-acid substitution or deletion of conserved structural motifs (nuclear localisation signal, polyproline motif, leucine zipper) and also affect exonic splicing enhancer motifs and physiological splice sites, suggesting potential deleterious effects on ATIP3 function and/or expression.