Expression and Functional Characterization of the Cancer-related Serine Protease, Human Tissue Kallikrein 14

Human tissue kallikrein 14 (KLK14) is a novel extracellular serine protease. Clinical data link KLK14 expression to several diseases, primarily cancer; however, little is known of its (patho)-physiological role. To functionally characterize KLK14, we expressed and purified recombinant KLK14 in mature and proenzyme forms and determined its expression pattern, specificity, regulation, and in vitro substrates. By using our novel immunoassay, the normal and/or diseased skin, breast, prostate, and ovary contained the highest concentration of KLK14. Serum KLK14 levels were significantly elevated in prostate cancer patients compared with healthy males. KLK14 displayed trypsin-like specificity with high selectivity for P1-Arg over Lys. KLK14 activity could be regulated as follows: 1) by autolytic cleavage leading to enzymatic inactivation; 2) by the inhibitory serpins alpha1-antitrypsin, alpha2-antiplasmin, antithrombin III, and alpha1-antichymotrypsin with second order rate constants (k(+2)/Ki) of 49.8, 23.8, 1.48, and 0.224 microM(-1) min(-1), respectively, as well as plasminogen activator inhibitor-1; and 3) by citrate and zinc ions, which exerted stimulatory and inhibitory effects on KLK14 activity, respectively. We also expanded the in vitro target repertoire of KLK14 to include collagens I-IV, fibronectin, laminin, kininogen, fibrinogen, plasminogen, vitronectin, and insulin-like growth factor-binding proteins 2 and 3. Our results indicate that KLK14 may be implicated in several facets of tumor progression, including growth, invasion, and angiogenesis, as well as in arthritic disease via deterioration of cartilage. These findings may have clinical implications for the management of cancer and other disorders in which KLK14 activity is elevated

VEGF regulates local inhibitory complement proteins in the eye and kidney

10.1172/JCI86418Journal of Clinical Investigation1271199-21

Synaptic proximity enables NMDAR signalling to promote brain metastasis.

Metastasis-the disseminated growth of tumours in distant organs-underlies cancer mortality. Breast-to-brain metastasis (B2BM) is a common and disruptive form of cancer and is prevalent in the aggressive basal-like subtype, but is also found at varying frequencies in all cancer subtypes. Previous studies revealed parameters of breast cancer metastasis to the brain, but its preference for this site remains an enigma. Here we show that B2BM cells co-opt a neuronal signalling pathway that was recently implicated in invasive tumour growth, involving activation by glutamate ligands of N-methyl-D-aspartate receptors (NMDARs), which is key in model systems for metastatic colonization of the brain and is associated with poor prognosis. Whereas NMDAR activation is autocrine in some primary tumour types, human and mouse B2BM cells express receptors but secrete insufficient glutamate to induce signalling, which is instead achieved by the formation of pseudo-tripartite synapses between cancer cells and glutamatergic neurons, presenting a rationale for brain metastasis.This work was principally supported by grants from the Swiss National Science Foundation and the European Research Council, and by a gift from the Biltema Foundation that was administered by the ISREC Foundation, Lausanne, Switzerland

<em>piggyBac</em> transposition reprograms fibroblasts to induced pluripotent stem cells

Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state. The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a spectrum of adult cell types. Current reprogramming strategies involve retroviral, lentiviral, adenoviral and plasmid transfection to deliver reprogramming factor transgenes. Although the latter two methods are transient and minimize the potential for insertion mutagenesis, they are currently limited by diminished reprogramming efficiencies. piggyBac (PB) transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines. The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events. Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition. Stable iPS cells thus generated express characteristic pluripotency markers and succeed in a series of rigorous differentiation assays. By taking advantage of the natural propensity of the PB system for seamless excision, we show that the individual PB insertions can be removed from established iPS cell lines, providing an invaluable tool for discovery. In addition, we have demonstrated the traceless removal of reprogramming factors joined with viral 2A sequences delivered by a single transposon from murine iPS lines. We anticipate that the unique properties of this virus-independent simplification of iPS cell production will accelerate this field further towards full exploration of the reprogramming process and future cell-based therapies. © 2009 Macmillan Publishers Limited. All rights reserved.Link_to_subscribed_fulltex

A set of microRNAs coordinately controls tumorigenesis, invasion, and metastasis

MicroRNA-mediated gene regulation has been implicated in various diseases, including cancer. This study examined the role of microRNAs (miRNAs) during tumorigenesis and malignant progression of pancreatic neuroendocrine tumors (PanNETs) in a genetically engineered mouse model. Previously, a set of miRNAs was observed to be specifically up-regulated in a highly invasive and metastatic subtype of mouse and human PanNET. Using functional assays, we now implicate different miRNAs in distinct phenotypes: miR-137 stimulates tumor growth and local invasion, whereas the miR-23b cluster enables metastasis. An algorithm, Bio-miRTa, has been developed to facilitate the identification of biologically relevant miRNA target genes and applied to these miRNAs. We show that a top-ranked miR-137 candidate gene, Sorl1, has a tumor suppressor function in primary PanNETs. Among the top targets for the miR-23b cluster, Acvr1c/ALK7 has recently been described to be a metastasis suppressor, and we establish herein that it is down-regulated by the miR-23b cluster, which is crucial for its prometastatic activity. Two other miR-23b targets, Robot and P2ryl, also have demonstrable antimetastatic effects. Finally, we have used the Bio-miRTa algorithm in reverse to identify candidate miRNAs that might regulate activin B, the principal ligand for ALK7, identifying thereby a third family of miRNAs-miRNA-130/301-that is congruently up-regulated concomitant with down-regulation of activin B during tumorigenesis, suggestive of functional involvement in evasion of the proapoptotic barrier. Thus, dynamic up-regulation of miRNAs during multistep tumorigenesis and malignant progression serves to down-regulate distinctive suppressor mechanisms of tumor growth, invasion, and metastasis

ALK7 Signaling Manifests a Homeostatic Tissue Barrier That Is Abrogated during Tumorigenesis and Metastasis

Herein, we report that the TGF beta superfamily receptor ALK7 is a suppressor of tumorigenesis and metastasis, as revealed by functional studies in mouse models of pancreatic neuroendocrine and lumina! breast cancer, complemented by experimental metastasis assays. Activation in neoplastic cells of the ALK7 signaling pathway by its principal ligand activin B induces apoptosis. During tumorigenesis, cancer cells use two different approaches to evade this barrier, either downregulating activin B and/or downregulating ALK7. Suppressing ALK7 expression additionally contributes to the capability for metastatic seeding. ALK7 is associated with shorter relapse-free survival of various human cancers and distant-metastasis-free survival of breast cancer patients. This study introduces mechanistic insights into primary and metastatic tumor development, in the form of a protective barrier that triggers apoptosis in cells that are not "authorized" to proliferate within a particular tissue, by virtue of those cells expressing ALK7 in a tissue microenvironment bathed in its ligand

KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome

<div><p>Netherton Syndrome (NS) is a rare and severe autosomal recessive skin disease which can be life-threatening in infants. The disease is characterized by extensive skin desquamation, inflammation, allergic manifestations and hair shaft defects. NS is caused by loss-of-function mutations in <i>SPINK5</i> encoding the LEKTI serine protease inhibitor. LEKTI deficiency results in unopposed activities of kallikrein-related peptidases (KLKs) and aberrantly increased proteolysis in the epidermis. <i>Spink5</i>^<i>-/-</i> mice recapitulate the NS phenotype, display enhanced epidermal Klk5 and Klk7 protease activities and die within a few hours after birth because of a severe skin barrier defect. However the contribution of these various proteases in the physiopathology remains to be determined. In this study, we developed a new murine model in which <i>Klk5</i> and <i>Spink5</i> were both knocked out to assess whether Klk5 deletion is sufficient to reverse the NS phenotype in <i>Spink5</i>^<i>-/-</i> mice. By repeated intercrossing between <i>Klk5</i>^<i>-/-</i> mice with <i>Spink5</i>^<i>-/-</i> mice, we generated <i>Spink5</i>^<i>-/-</i><i>Klk5</i>^<i>-/-</i> animals. We showed that <i>Klk5</i> knock-out in Lekti-deficient newborn mice rescues neonatal lethality, reverses the severe skin barrier defect, restores epidermal structure and prevents skin inflammation. Specifically, using <i>in situ</i> zymography and specific protease substrates, we showed that <i>Klk5</i> knockout reduced epidermal proteolytic activity, particularly its downstream targets proteases KLK7, KLK14 and ELA2. By immunostaining, western blot, histology and electron microscopy analyses, we provide evidence that desmosomes and corneodesmosomes remain intact and that epidermal differentiation is restored in <i>Spink5</i>^<i>-/-</i><i>Klk5</i>^<i>-/-</i>. Quantitative RT-PCR analyses and immunostainings revealed absence of inflammation and allergy in <i>Spink5</i>^<i>-/-</i><i>Klk5</i>^<i>-/-</i> skin. Notably, Il-1β, Il17A and Tslp levels were normalized. Our results provide <i>in vivo</i> evidence that KLK5 knockout is sufficient to reverse NS-like symptoms manifested in <i>Spink5</i>^<i>-/-</i> skin. These findings illustrate the crucial role of protease regulation in skin homeostasis and inflammation, and establish KLK5 inhibition as a major therapeutic target for NS.</p></div

Loss of <i>Klk5</i> expression reverses skin and whisker anomalies in <i>Spink5</i>^<i>-/-</i> mice.

<p>(<b>A</b>) Macroscopic appearance of wt, <i>Spink5</i>^<i>-/-</i>, <i>Klk5</i>^<i>-/-</i>, <i>Spink5</i>^<i>-/-</i><i>Klk5</i>^<i>-/-</i> mice. Photos were taken 30 h after birth for wt, <i>Klk5</i>^<i>-/-</i> and <i>Spink5</i>^<i>-/-</i><i>Klk5</i>^<i>-/-</i> mice and 5 h for <i>Spink5</i>^<i>-/-</i> mice; (<b>B</b>) Microscopic appearance of muzzle area (scale bar 1mm) and (<b>C</b>) whisker ultrastructure by scanning electron microscopy (SEM). Scale bar, 10μm.</p

Klk5 is a key regulator of epidermal proteolysis.

<p><i>In situ</i> zymography using fluorescence-quenched casein (<b>A)</b> or elastin (<b>B</b>). Skin tissue sections were prepared from wt, <i>Spink5</i>^<i>-/-</i>, <i>Klk5</i>^<i>-/-</i>, and <i>Spink5</i>^<i>-/-</i><i>Klk5</i>^<i>-/-</i> mice. Ablation of <i>Klk5</i> expression highly reduced both caseinolytic and elastinolytic activities in the <i>stratum corneum</i> of <i>Spink5</i>^<i>-/-</i><i>Klk5</i>^<i>-/-</i> as compared to <i>Spink5</i>^<i>-/-</i>, respectively. Fluorescence intensity data was transformed into a color gradient (as shown) using ImageJ software. Dashed white lines represent the dermal-epidermal junction; <u>The asterisk (*) indicates the epidermis, scale bar: 25μm</u> (<b>C</b>) Changes in proteolytic activity using colorimetric substrates that target different proteases. Activity in wt, <i>Spink5</i>^<i>-/-</i> and <i>Spink5</i>^<i>-/-</i><i>Klk5</i>^<i>-/-</i> was detected by measuring absorbance at 405 nm after overnight incubation with substrates for either KLK7 or KLK14. Data are shown as the mean ± SEM of duplicates for four mice per genotype; (<b>D</b>) Analysis of mRNA expression in skin by RT-qPCR of Klk7 and Klk14. Results show high expression of both Klks in <i>Spink5</i>^<i>-/-</i>. In <i>Spink5</i>^<i>-/-</i><i>Klk5</i>^<i>-/-</i> skin, expression of Klk14 is equal to wt while Klk7 remained slightly higher. Data are shown as the mean ± s.e.m. of triplicate amplification for at least three mice per genotype. Results are normalized to wt mean (set as 1.0).</p