A case-only study to identify genetic modifiers of breast cancer risk for BRCA1/BRCA2 mutation carriers.

Breast cancer (BC) risk for BRCA1 and BRCA2 mutation carriers varies by genetic and familial factors. About 50 common variants have been shown to modify BC risk for mutation carriers. All but three, were identified in general population studies. Other mutation carrier-specific susceptibility variants may exist but studies of mutation carriers have so far been underpowered. We conduct a novel case-only genome-wide association study comparing genotype frequencies between 60,212 general population BC cases and 13,007 cases with BRCA1 or BRCA2 mutations. We identify robust novel associations for 2 variants with BC for BRCA1 and 3 for BRCA2 mutation carriers, P < 10-8, at 5 loci, which are not associated with risk in the general population. They include rs60882887 at 11p11.2 where MADD, SP11 and EIF1, genes previously implicated in BC biology, are predicted as potential targets. These findings will contribute towards customising BC polygenic risk scores for BRCA1 and BRCA2 mutation carriers

Phosphodiesterase activity and specificity measured using microcalorimetry

Neben den cAMP- und cGMP-abhängigen Proteinkinasen (PKA bzw. PKG) sind als zyklonukleotid-regulierte Effektorproteine die Ionenkanäle CNG1-4, der "guanine nucleotide exhange factor“ Epac sowie die zyklonukleotid-spaltende Familie der Phosphodiesterasen (PDEs) von Bedeutung. Industriell synthetisierte cGMP- und cAMP-Analoga besitzen zwar meist eine hohe Affinität für ihr Zielprotein, über ihre Hydrolysestabilität gegenüber PDEs in der Zelle ist jedoch wenig bekannt. In dieser Arbeit wurden die kinetischen Konstanten von elf der am häufigsten genutzten cAMP-und cGMP-Analoga an verschiedenen Vertretern der PDE-Familien mittels Mikrokaloriemetrie bestimmt. Zudem konnte in den Messungen der inhibitorisch Effekt hydrolysestabiler Derivate auf die PDEs qualitativ und quantitativ ermittelt werden kann. Die Ergebnisse zeigen, dass Phosphodiesterasen in der Lage sind, auch chemisch modifizierte Analogsubstanzen der Cyclonukleotide cAMP und cGMP zu hydrolysieren. Hydrolysestabile Derivate dagegen entwickeln häufig inhibitorische Wirkung auf die PDEs und verursachen dadurch Veränderungen der intrazellulären cAMP und cGMP Konzentrationen. So vermag z. B. die Epac-spezifische Substanz Sp-8-pCPT-2’-O-Me-cAMPS in den in vitro Experimenten die PDEs mit ki-Werten im einstelligen mikromolaren Bereich zu inhibieren. In mit Sp-8-pCPT-2’-O-Me-cAMPS stimulierten Thrombozyten steigt als Folge dieser PDE-Hemmung die cGMP Konzentration in der Zelle an und man beobachtet eine als Folge eine PKG-vermittelte Phosphorylierung des Substratproteins VASP – eine unerwünschte Nebenreaktion. Die erhobenen Daten lassen außerdem Rückschlüsse auf den Inhibitionsmechanismus zu. Einige Analoga inhibieren die cGMP-bindenden GAF-Domänen in den PDEs 2A, 5A, 6cone und 10A sowie die PDE 4D3 nach dem linear-mixed-Typ und beeinflussen daher, neben der katalytischen Aktivität, vermutlich auch regulatorische Zentren dieser Enzyme. Zusammenfassend erleichtern die erhobenen Daten Wissenschaftlern die Auswahl des für ihre Fragestellung am besten geeigneten Derivates.cAMP and cGMP are critical second messengers that regulate multiple targets including different cAMP/cGMP-dependent protein kinases (PKA/PKGs), exchange proteins directly activated by cAMP (Epacs), phosphodiesterases (PDEs) and cyclic nucleotide-gated ion channels (CNGs). Second and third generation cyclic nucleotide analogs are widely used to elucidate specificity of cellular signaling, mediated by these target proteins. However, the selectivity and stability of these analogs need to be fully understood in order to properly interpret results and rigorously assess the mechanisms by which these analogs work in the cell. To better understand the selectivity and cross-reactivity of these analogs I measured the activation or inhibitory activity of 13 commonly-used cyclic nucleotide analogs 8 different PDEs. To measure their stability to hydrolysis I utilized isothermal microcalorimetry, a method that allows to evaluate whether or not an analog can function as a substrate or inhibitor for PDEs. I demonstrate that indeed some of these analogs can be hydrolyzed by multiple PDEs and others are competitive inhibitors. Herein I provide Ki data for all of the non-hydrolyzable analogs and Km and Vmax values for all of the hydrolyzable analogs. Each of these values, as well as their mode of inhibition can be determined in a single experiment. The data strongly implied that several of these analogs might, in addition to their primary effects, also cause elevation of cAMP or cGMP indirectly by inhibiting PDEs in the cell. Such an effect could of course cloud interpretation of the use of these analogs. Similarly, those that are PDE substrates also might have their duration of action substantially reduced. To illustrate this point we show that Sp-8-pCPT-2’-O-Me-cAMPS, a highly specific non-hydrolyzable Epac activator in vitro, can under certain conditions enhance cGMP/PKG and cAMP/PKA signaling pathways in intact platelets. Specifically we found enhanced VASP phosphorylation at both PKA and PKG phosphorylation sites after the addition of Sp-8-pCPT-2’-O-Me-cAMPS. These data indicate that this “selective Epac activator” is able to indirectly activate the cAMP/PKA and cGMP/PKG signalling pathways presumably through inhibition of platelet PDE5 and/or PDE3. The data together allow to provide recommendations for how best to probe the different cyclic nucleotide signalling pathways using cyclic nucleotide analogs. In summary, the data provide evidence that most cAMP and cGMP analogs have multiple targets. Therefore, interpretation of any effects these analogs have in cells should take into consideration their possible cross-target reactivities

LASP1, a Newly Identified Melanocytic Protein with a Possible Role in Melanin Release, but Not in Melanoma Progression

The LIM and SH3 protein 1 (LASP1) is a focal adhesion protein. Its expression is increased in many malignant tumors. However, little is known about the physiological role of the protein. In the present study, we investigated the expression and function of LASP1 in normal skin, melanocytic nevi and malignant melanoma. In normal skin, a distinct LASP1 expression is visible only in the basal epidermal layer while in nevi LASP1 protein is detected in all melanocytes. Melanoma exhibit no increase in LASP1 mRNA compared to normal skin. In melanocytes, the protein is bound to dynamin and mainly localized at late melanosomes along the edges and at the tips of the cell. Knockdown of LASP1 results in increased melanin concentration in the cells. Collectively, we identified LASP1 as a hitherto unknown protein in melanocytes and as novel partner of dynamin in the physiological process of membrane constriction and melanosome vesicle release

Correlation of LASP1 expression to clinicopathological parameters in melanoma.

<p>* Tumor depth after AJCC guidelines.</p><p>** within 5 years of follow-up. Values of <i>p</i> were calculated by Chi-Square test; statistical significance is assumed when p<0.05.</p><p>Correlation of LASP1 expression to clinicopathological parameters in melanoma.</p

LASP1 expression in samples of melanocytic nevi, primary melanomas and melanoma metastases.

<p>LASP1 expression in samples of melanocytic nevi, primary melanomas and melanoma metastases.</p

Immunofluorescence of LASP1 and tyrosinase in NHEM cells.

<p>Black and white image of a NHEM cell with highlighted blue nucleus and circled enlarged dendrite tip (upper panel). LASP1 (red) and tyrosinase (green) immunofluorescence at the dendrite tip of the NHEM cell (middle panels). Co-localization of the merged LASP1-tyrosinase complex (white arrows) with pointed melanosomes (black arrows) in the bright-field image at the dendrite tip of the NHEM cell (lower panels).</p

Proposed model for LASP1 involvement in actin-dynamin- mediated melanosome vesicle scission at melanocyte dendrite tips.

<p>In anterograde melanosomal transport mature melanosomes move along microtubules by means of the motor protein kinesin and are transferred towards the cell periphery. Once at the periphery, track switching from microtubules to actin filaments occurs, a process mediated by Rab27A and molecular motor protein myosin Va (1). In this fashion microtubule, actin filaments and motor systems co-operate to promote melanosome transport and retention of the organelle in peripheral dendrites. LASP1 and dynamin are present at the actin mesh at the plasma membrane. LASP1 binds to actin and dynamin through its nebulin repeat and SH3 domain, respectively. Dynamin exists as a dimer and binds to actin through the stalk region (2). WASP- and CDC42-mediated activation of ARP2/3 lead to the branched polymerization of actin filaments towards the release site. Actin, together with other motor proteins, pushes the plasma membrane and enhances membrane invagination (3). As an adaptor/scaffolding protein, LASP1 recruits and positions dynamin at the tubular membrane (4). Subsequent polymerization of dynamin around the membrane in a helical manner and GTP hydrolysis results in membrane constriction and melanosome vesicle scission at dendrite tips (5 and 6).</p

LASP1 expression in normal skin and nevi.

<p>(A) Representative immunohistochemical staining of consecutive sections from normal skin and from a representative melanocytic nevus for LASP1 and the melanocytic antigen MART1 (magnification 20x; Scale bar = 100 μm). Stratum basale of normal skin and nevus shows distinct LASP1-positivity. Histiocytes (yellow arrows) and a transverse cut blood vessel (red arrow) in the dermis display LASP1 expression. (B) Immunohistochemical LASP1 staining of normal skin and nevus at higher magnification (100x; Scale bar = 10 μm). Nuclear LASP1-positive cells of the epidermal basal layer in normal skin are marked with arrows. Hematoxylin is used for counterstaining.</p

Nuclear and cytosolic LASP1 distribution.

<p>(A) Western blot analysis of LASP1 and pLASP1-S146 in cytosolic (C) and nuclear (N) fractions of MaMel2, UACC257, normal human epidermal melanocytes (NHEM) and breast cancer cell line MDAMB231 before and after forskolin stimulation. Phosphorylation of LASP1 by PKA does not influence cytosolic LASP1 localization in melanoma cell lines and NHEM. Purity and loading of the fractions were controlled by Western blot for β-actin, the cytosolic marker GAPDH and the nuclear markers Lamin A/C and histone H2B. (B) Western blot analysis of LASP1, ZO2 and PKA expression in MDA-MB 231 breast cancer cells compared to melanoma cell lines and NHEM. Loading was adjusted to similar β-actin levels.</p

Co-localization of LASP1 and dynamin in melanosomes.

<p>(A) MaMel2 cells were prepared as described in “Materials and Methods” and loaded on a sucrose density-gradient. Equal fraction volumes were subjected to SDS/PAGE and immunoblotted for LASP1, TRP1, tyrosinase and dynamin. “Original before” indicates the loading sample in 0.25 M sucrose before centrifugation. “Original after” indicates the loading sample fraction after centrifugation. The protein concentrations of the fractions and the corresponding melanosome stages are stated. (B) Sucrose density gradient tube with concentrated melanosomes (indicated by black arrows) in the 1.8 M sucrose gradient fraction. (C) LASP1 (red) and dynamin (green) immunofluorescence at the dendrite tip of a MaMel2 cell. Co-localization of the merged LASP1-dynamin complex (yellow) with melanosomes (black arrows) in the bright-field image at the dendrite tip of the MaMel2 cell.</p