Sébastien Jeay

ARQ 197, a Novel and Selective Inhibitor of the Human c-Met Receptor Tyrosine Kinase with Antitumor Activity

The met proto-oncogene is functionally linked with tumorigenesis and metastatic progression. Validation of the receptor tyrosine kinase c-Met as a selective anticancer target has awaited the emergence of selective c-Met inhibitors. Herein, we report ARQ 197 as the first non-ATP–competitive small molecule that selectively targets the c-Met receptor tyrosine kinase. Exposure to ARQ 197 resulted in the inhibition of proliferation of c-Met–expressing cancer cell lines as well as the induction of caspase-dependent apoptosis in cell lines with constitutive c-Met activity. These cellular responses to ARQ 197 were phenocopied by RNAi-mediated c-Met depletion and further demonstrated by the growth inhibition of human tumors following oral administration of ARQ 197 in multiple mouse xenograft efficacy studies. Cumulatively, these data suggest that ARQ 197, currently in phase II clinical trials, is a promising agent for targeting cancers in which c-Met-driven signaling is important for their survival and proliferation. Mol Cancer Ther; 9(6); 1544–53. ©2010 AACR.

Growth hormone can act as a cytokine controlling survival and proliferation of immune cells: new insights into signaling pathways

While growth hormone (GH) is classically defined as a peptide hormone, recent evidence supports a role for GH acting as a cytokine in the immune system under conditions of stress, counteracting immunosuppression by glucocorticoids. Lymphoid cells express the GH receptor, which belongs to the cytokine receptor superfamily, and GH can be produced by immune tissues, suggesting an autocrine/paracrine mode of action of GH. GH can act as a cytokine, promoting cell cycle progression of lymphoid cells and preventing apoptosis. These effects of GH were shown to be mainly mediated by the PI-3 kinase/Akt pathway and the transcription factor NF-kappaB. Expression of several cell cycle mediators, as well as Bcl-2, c-Myc and cyclin proteins were found to be regulated by GH. Survival of immune cells under conditions of stress was promoted by NF-kappaB. Thus, GH acts not only as a hormone but also as a cytokine, playing a potentially important role in immune system cells. Lastly, in this mini-review, we will discuss whether the discovery of these molecules in GH signaling pathways offers new insights into additional mechanisms of action whereby GH regulates apoptosis, proliferation and neoplastic transformation of cells of the immune system.

Human prolactin (hPRL) antagonists inhibit hPRL-activated signaling pathways involved in breast cancer cell proliferation.

The involvement of human prolactin (hPRL) in breast cancer has been recently reconsidered based on its autocrine/paracrine proliferative effect described in human mammary tumor epithelial cells. Therefore, there is growing interest in the development of potent hPRL antagonists that may inhibit this effect. We previously designed hPRL analogs displaying antagonistic properties in a human transcriptional bioassay. We now report that the most potent of those analogs, G129R-hPRL, antagonizes all hPRL-induced effects analysed in various breast cancer cell lines, including cell proliferation. The analog per se lacks intrinsic agonistic activity on PRL receptor-activated signaling cascades, cell proliferation and apoptosis, indicating that its mode of action only occurs through competitive inhibition of hPRL. We provide some molecular basis of this antagonistic effect by demonstrating that G129R-hPRL competitively inhibits hPRL-activation of the JAK-STAT and MAPK pathways, two signaling cascades involved in the mitogenic effect of hPRL in mammary epithelial cells. This competitive inhibition persists for at least 48 h, as evidenced by long term analysis of STAT5b activation or of progression through cell cycle. These results are the first demonstration at the molecular level that hPRL antagonists interfering with receptor dimerization disrupt signaling events in breast cancer cells, which prevents hPRL-induced cell proliferation.

The Tumor Suppressor Activity of the Lysyl Oxidase Propeptide Reverses the Invasive Phenotype of Her-2/neu–Driven Breast Cancer

Expression of the lysyl oxidase gene (LOX) was found to inhibit the transforming activity of the ras oncogene in NIH 3T3 fibroblasts and was hence named the ras recision gene (rrg). Lysyl oxidase (LOX) is synthesized and secreted as a 50-kDa inactive proenzyme (Pro-LOX), which is processed by proteolytic cleavage to a functional 32-kDa enzyme and an 18-kDa propeptide (LOX-PP). Recently, the ras recision activity of the LOX gene in NIH 3T3 cells was mapped to its propeptide region. Here, we show for the first time that LOX-PP inhibits transformation of breast cancer cells driven by Her-2/neu, an upstream activator of Ras. LOX-PP expression in Her-2/neu-driven breast cancer cells in culture suppressed Akt, extracellular signal-regulated kinase, and nuclear factor-kappaB activation. Her-2/neu-induced epithelial to mesenchymal transition was reverted by LOX-PP, as judged by reduced levels of Snail and vimentin; up-regulation of E-cadherin, gamma-catenin, and estrogen receptor alpha; and decreased ability to migrate or to form branching colonies in Matrigel. Furthermore, LOX-PP inhibited Her-2/neu tumor formation in a nude mouse xenograft model. Thus, LOX-PP inhibits signaling cascades induced by Her-2/neu that promote a more invasive phenotype and may provide a novel avenue for treatment of Her-2/neu-driven breast carcinomas.

Reciprocal Control of Forkhead box O 3a and c-Myc via the Phosphatidylinositol 3-Kinase Pathway Coordinately Regulates p27Kip1 Levels

B cell receptor (BCR) engagement of murine WEHI 231 immature B lymphoma cells leads sequentially to a drop in NF-κB and c-Myc, and induction of the p27Kip1 cyclin-dependent kinase inhibitor, which promotes growth arrest and apoptosis. BCR engagement was recently shown to induce a drop in phosphatidylinositol 3-kinase (PI3K)/Akt signaling, preceding the increase in p27. As induction of p27 is due to an increase in gene transcription, we investigated the role of the Forkhead box O (FOXO) transcription factor family, which has been shown to potently induce p27 promoter activity. We demonstrate that pharmacologic inhibitors of PI3K or BCR engagement lead to decreased inactive cytoplasmic levels and increased active functional nuclear FOXO3a. In contrast, inhibition of PI3K/Akt signaling decreased the levels of NF-κB and c-Myc, which has been shown to repress p27 promoter activity. To test the effects of ectopic c-Myc on endogenous p27 levels, WEHI 231 cells stably expressing c-Myc or empty vector DNA were prepared. Ectopic c-Myc blocked the induction of p27 expression upon either inhibition of PI3K or BCR engagement. Thus, p27Kip1 is coordinately regulated via two arms of a signaling pathway that are inversely controlled upon inhibition of PI3K: induction of the activator FOXO3a and down-regulation of the repressor c-Myc.

The Public Repository of Xenografts Enables Discovery and Randomized Phase II-like Trials in Mice

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease.

Growth Hormone Exerts Antiapoptotic and Proliferative Effects through Two Different Pathways Involving Nuclear Factor-κB and Phosphatidylinositol 3-Kinase1

Dependence of murine pro-B Ba/F3 cells on interleukin-3 can be substituted by GH when cells are stably transfected with the GH receptor (GHR) complementary DNA. Recently, we demonstrated that Ba/F3 cells produce GH, which is responsible for the survival of cells expressing the GHR. This GH effect involves the activation of nuclear factor-kB (NF-kB). Here, we examined the signaling pathways mediating proliferation of growth factor-deprived Ba/F3 GHR cells. Exogenous GH stimulation of Ba/F3 GHR cells induced cyclins E and A and the cyclin-dependent kinase inhibitor p21 and repressed cyclin-dependent kinase inhibitor p27. The presence of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor Ly 294002 abolished proliferation induced by GH, arresting Ba/F3 GHR cells at the G1/S boundary, but did not promote apoptosis. Thus, the proliferative effect of GH is closely related to PI 3-kinase activation, whereas PI 3-kinase is not essential for GH-induced cell survival. Addition of Ly 294002 resulted in a moderate decrease in NF-kB activation by GH, suggesting a possible link between PI 3-kinase and NF-kB signaling by GH. Expression of c-myc was also induced by GH in Ba/F3 GHR cells, and inactivation of either PI 3-kinase or NF-kB reduced this induction. Overexpression of the dominant negative repressor mutant c-Myc-RX resulted in an inhibition of the GH proliferative effect, suggesting the involvement of c-myc in GH-induced proliferation. Taken together, these results suggest that the effects of GH on cell survival and proliferation are mediated through two different signaling pathways, NF-kB and PI 3-kinase, respectively; although cross-talk between them has not been excluded. NF-kB, which has been shown to be responsible for the antiapoptotic effect of GH, could also participate in GH-induced proliferation, as c-myc expression is promoted by PI 3-kinase, in an NF-kB-dependent and -independent manner. (Endocrinology 142: 147–156, 2001) G INITIATES its wide variety of biological effects by binding to the GH receptor (GHR). After GHR dimerization, Jak-2 recruitment and activation promote its autophosphorylation and phosphorylation of GHR on tyrosine residues. In addition, a large number of other cellular proteins become tyrosyl phosphorylated in response to GH (1). Two signaling pathways have been identified to mediate the GH proliferative action: the mitogen-activated protein (MAP) kinases, designated extracellular regulated kinase-1 (Erk-1) and Erk-2, and signal transducers and activators of transcription. Molecules implicated in MAP kinase activation by GH were identified in 293 cells (2), and their involvement was confirmed in 3T3 F442A preadipocytes (3). The Shc/Grb2/SOS/Ras/Raf/MEK (MAP/Erk kinase)/MAP kinase pathway activated by GH led to the activation of several transcription factors, such as Elk-1, a member of the ternary complex factor family. GH induces phosphorylation of Elk-1 via Erk-1 and -2 and promotes the transcription of early response genes such as c-fos, egr-1, and junB (4). Furthermore, a correlation was demonstrated between insulin receptor substrate-1 (IRS-1) expression and GH-induced MAP kinase activation and proliferation in GHR-expressing 32D cells (5). Together, these data strongly suggest an important role for the MAP kinase pathway in the GH prolif-

New homologous bioassays for human lactogens show that agonism or antagonism of various analogs is a function of assay sensitivity

The reference bioassay for lactogens is the Nb2 cell proliferation assay, whose extreme sensitivity allows the detection of very low amounts of lactogenic activity in biologic fluids. The use of rat Nb2 cells raises the problem of species specificity when analyzing lactogens of other origin, including human lactogenic hormones for which no reference bioassay currently exists. In this article, we describe two new homologous bioassays for human lactogens. One is a transcriptional bioassay generated by stably transfecting 293 human embryonic kidney fibroblasts using two plasmids, encoding the human prolactin receptor (hPRLR) and the PRL-responsive lactogenic hormone response element-luciferase reporter gene. The second is a proliferation assay obtained by stably transfecting Ba/F3 cells with a plasmid encoding the hPRLR. We provide characterization of the various clones or cell populations that were isolated, and we describe experiments that were performed to achieve optimized protocols for both bioassays. These new assays were compared with other cells types exhibiting well-recognized PRL-mediated responses (proliferation of Nb2 or of human breast tumor cell lines), using various lactogen analogs. This comparative analysis provides strong evidence that the intrinsic characteristics of each bioassay dramatically affect the biologic properties attributed to the lactogen of interest. Depending on the assay, a given analog can exhibit agonistic or antagonistic properties. We hypothesize that in addition to species specificity, assay sensitivity is the key parameter in directing the apparent bioactivity of lactogens. Of course, in the end, it will be necessary to confirm the agonistic or antagonistic properties of the tested analogs, in vivo.

The Proliferative and Antiapoptotic Actions of Growth Hormone and Insulin-Like Growth Factor-1 Are Mediated through Distinct Signaling Pathways in the Pro-B Ba/F3 Cell Line1

Biological actions of GH can be direct or mediated through insulin-like growth factor I (IGF-I). In the interleukin-3 (IL-3)-dependent Ba/F3 cell line, IGF-I induces cell cycle entry and proliferation. Ba/F3 cells expressing the rat GH receptor (Ba/F3 GHR cells) have been shown to escape from apoptosis and to proliferate under GH stimulation. Using the Ba/F3 GHR cell model, we sought to dissect the signals elicited specifically by IGF-I or GH. In contrast to IGF-I or IL-3, GH is able to maintain cell cycle entry of Ba/F3 GHR cells cultured for 7 days in the absence of serum. The presence of IGF-I messenger RNA was not detected by RT-PCR, and by RIA, IGF-I was not found in culture medium of Ba/F3 GHR cells, unstimulated or stimulated by GH. Moreover, the addition of an anti-IGF-I antibody that blocks IGF-I effects suggests that the actions of GH are not mediated by IGF-I, but appear to be direct. GH or IGF-I stimulation increased expression of cyclins A and D(1) with comparable kinetics, whereas expression of p21(waf1/cip1) seemed delayed in IGF-I-stimulated cells compared with that in GH-stimulated cells. Contrary to GH or IL-3, IGF-I did not induce nuclear factor-kappaB DNA-binding activity in Ba/F3 cells. Inhibition of nuclear factor-kappaB through expression of the mutant IkappaBalpha (A32/36) abrogated the GH-mediated survival signal, but did not result in alterations of the cell cycle in Ba/F3 GHR cells treated with IGF-I. Phosphatidylinositol 3-kinase was required for both survival and proliferative responses to IGF-I. Transfection of a dominant negative form of AKT (AH-AKT) resulted in suppression of IGF-I-mediated cell survival, but not of the antiapoptotic effect of GH in Ba/F3 GHR cells. Thus, GH and IGF-I are able to promote cell survival and proliferation through independent and different pathways in Ba/F3 cells.

c‐Myc represses FOXO3a‐mediated transcription of the gene encoding the p27Kip1 cyclin dependent kinase inhibitor

The p27Kip1 (p27) cyclin‐dependent kinase inhibitor and c‐Myc oncoprotein play essential roles in control of cell cycle progression and apoptosis. Induction of p27 (CDKN1B) gene transcription by Forkhead box O proteins such as FOXO3a leads to growth arrest and apoptosis. Previously, we observed that B cell receptor (surface IgM) engagement of WEHI 231 immature B lymphoma cells with an anti‐IgM antibody results in activation of FOXO3a, growth arrest and apoptosis. As ectopic c‐Myc expression in these cells prevented anti‐IgM induction of p27 and cell death, we hypothesized that c‐Myc represses FOXO3a‐mediated transcription. Here we show that c‐Myc inhibits FOXO3a‐mediated activation of the p27 promoter in multiple cell lines. The mechanism of this repression was explored using a combination of co‐immunoprecipitation, oligonucleotide precipitation, and chromatin immunoprecipitation experiments. The studies demonstrate a functional association of FOXO3a and c‐Myc on a proximal Forkhead binding element in the p27 promoter. This association involves the Myc box II domain of c‐Myc and the N‐terminal DNA‐binding portion of FOXO3a. Analysis of publicly available microarray datasets showed an inverse pattern of c‐MYC and p27 RNA expression in primary acute myeloid leukemia, prostate cancer and tongue squamous cell carcinoma samples. The inhibition of FOXO3a‐mediated activation of the p27 gene by the high aberrant expression of c‐Myc in many tumor cells likely contributes to their uncontrolled proliferation and invasive phenotype. J. Cell. Biochem. 104: 2091–2106, 2008.