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Identification of early cancer, detection of progression, and monitoring of therapeutic success remain urgent issues in clinical medicine, particularly given the increasing cancer incidence in our aging populations. New methodologies have provided enormous progress over the past decades by defining the genetic and proteomic composition of cancers, yielding putative cancer biomarkers detectable in blood or other body fluids less invasively and more cheaply than using currently available screening techniques that often involve biopsies or surgery. However, the clinical use of these new methodologies is still far off. In this review, we focus on putative soluble cancer biomarkers shed from the cell surface by metalloproteases overexpressed in numerous cancers. Although useful candidates have been identified, their validation and adoption into clinical use remain challenging.

The immunomodulatory capacity of mesenchymal stem cells (MSCs) is critical for their use in therapeutic applications. MSC response to specific inflammatory cues allows them to switch between a proinflammatory (MSC1) or anti‐inflammatory (MSC2) phenotype. Regulatory mechanisms controlling this switch remain to be defined. One characteristic feature of MSC2 is their ability to respond to IFNγ with induction of indoleamine 2,3‐dioxygenase (IDO), representing the key immunoregulatory molecule released by human MSC. Here, we show that STAT1 and PI3Kα pathways interplay regulates IFNγ‐induced IDO production in MSC. Chemical phosphoinositide 3‐kinase (PI3K) pan‐inhibition, PI3Kα‐specific inhibition or shRNA knockdown diminished IFNγ‐induced IDO production. This effect involved PI3Kα‐mediated upregulation of STAT1 protein levels and phosphorylation at Ser727. Overexpression of STAT1 or of a constitutively active PI3Kα mutant failed to induce basal IDO production, but shifted MSC into an MSC2‐like phenotype by strongly enhancing IDO production in response to IFNγ as compared to controls. STAT1 overexpression strongly enhanced MSC‐mediated T‐cell suppression. The same effect could be induced using short‐term pretreatment of MSC with a chemical inhibitor of the counter player of PI3K, phosphatase and tensin homolog. Finally, downregulation of STAT1 abrogated the immunosuppressive capacity of MSC. Our results for the first time identify critical upstream signals for the induced production of IDO in MSCs that could be manipulated therapeutically to enhance their immunosuppressive phenotype. StemCells2015;33:1892–1901

Gonadotropin releasing hormone (GnRH) contributes to the maintenance of gonadotrope function by increasing extracellular signal-regulated kinase (ERK) activity subsequent to binding to its cognate G-protein-coupled receptor. As the GnRH receptor exclusively interacts with Gq/11proteins and as receptor expression is regulated in a β-arrestin-independent fashion, it represents a good model to systematically dissect underlying signaling pathways. In αT3-1 gonadotropes endogenously expressing the GnRH receptor, GnRH challenge resulted in a rapid increase in ERK activity which was attenuated by the epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitor AG1478. In COS-7 cells transiently expressing the human GnRH receptor, agonist-induced ERK activation was independent of free Gβγ subunits but could be mimicked by short-term phorbol ester treatment. Most notably, Gq/11-induced ERK activation was sensitive to N17-Ras and to expression of the C-terminal Src kinase but also to other dominant negative mutants of signaling components localized upstream of Ras, like Shc and the EGFR. GnRH as well as phorbol esters led to Ras activation in COS-7 and αT3-1 cells, which was dependent on Src and EGFR tyrosine kinases, indicating that both tyrosine kinases act downstream of protein kinase C (PKC) and upstream of Ras. However, Src did not contribute to Shc tyrosine phosphorylation. GnRH or phorbol ester challenge resulted in PKC-dependent EGFR autophosphorylation. Furthermore, a 5-min phorbol ester treatment was sufficient to trigger tyrosine phosphorylation of the platelet-derived growth factor-β receptor in L cells. Thus, in several cell systems PKC is able to stimulate Ras via activation of receptor tyrosine kinases.

The agonist-bound gonadotropin-releasing hormone (GnRH) receptor engages several distinct signaling cascades, and it has recently been proposed that coupling of a single type of receptor to multiple G proteins (Gq, Gs, and Gi) is responsible for this behavior. GnRH-dependent signaling was studied in gonadotropic αT3–1 cells endogenously expressing the murine receptor and in CHO-K1 (CHO#3) and COS-7 cells transfected with the human GnRH receptor cDNA. In all cell systems studied, GnRH-induced phospholipase C activation and Ca2+mobilization was pertussis toxin-insensitive, as was GnRH-mediated extracellular signal-regulated kinase activation. Whereas the Gi-coupled m2 muscarinic receptor interacted with a chimeric Gsprotein (Gsi5) containing the C-terminal five amino acids of Gαi2, the human GnRH receptor was unable to activate the G protein chimera. GnRH challenge of αT3–1, CHO#3 and of GnRH receptor-expressing COS-7 cells did not result in agonist-dependent cAMP formation. GnRH challenge of CHO#3 cells expressing a cAMP-responsive element-driven firefly luciferase did not result in increased reporter gene expression. However, coexpression of the human GnRH receptor and adenylyl cyclase I in COS-7 cells led to clearly discernible GnRH-dependent cAMP formation subsequent to GnRH-elicited rises in [Ca2+]i. In αT3–1 and CHO#3 cell membranes, addition of [α-32P]GTP azidoanilide resulted in GnRH receptor-dependent labeling of Gαq/11but not of Gαi, Gαsor Gα12/13proteins. Thus, the murine and human GnRH receptors exclusively couple to G proteins of the Gq/11family. Multiple GnRH-dependent signaling pathways are therefore initiated downstream of the receptor/G protein interface and are not indicative of a multiple G protein coupling potential of the GnRH receptor.

Binding of lutropin/choriogonadotropin to its cognate receptor results in the activation of adenylyl cyclase and phospholipase C. The mechanism underlying the generation of this bifurcating signal is presently not known. To analyze the coupling mechanism of the LH receptor, activated G proteins were labeled with [α-32P]GTP azidoanilide and identified by selective immunoprecipitation. In membranes of bovine corpora lutea and of L cells stably expressing the murine LH receptor (LHR cells), human chorionic gonadotropin (hCG) led to incorporation of the label into αsand αi2. Stimulation of LHR cells or of L cells expressing the M5muscarinic receptor (LM5 cells) with the respective agonist resulted in activation of phospholipase C in both cell lines. However, αqand α11were only labeled upon stimulation of the M5muscarinic receptor. Agonist-induced Ca2+mobilization and inositol phosphate accumulation were partially sensitive to pertussis toxin, and the expression of the βγ-stimulable phospholipase C isoforms β2 and β3 could be demonstrated in LHR cells. Overexpression of phospholipase C-β2 led to increased hCG-stimulated inositol phosphate accumulation, and expression of a β-ARK1 C-terminal polypeptide effectively suppressed hCG-mediated phosphatidylinositol hydrolysis. Thus, the LH receptor couples to both Gsand Gi, and βγ-subunits released from either G protein contribute to the stimulation of phospholipase C-β isoforms.