RasGRPs are targets of the anti-cancer agent ingenol-3-angelate.

Ingenol-3-angelate (I3A) is a non-tumor promoting phorbol ester-like compound identified in the sap of Euphoria peplus. Similar to tumor promoting phorbol esters, I3A is a diacylglycerol (DAG) analogue that binds with high affinity to the C1 domains of PKCs, recruits PKCs to cellular membranes and promotes enzyme activation. Numerous anti-cancer activities have been attributed to I3A and ascribed to I3A's effects on PKCs. We show here that I3A also binds to and activates members of the RasGRP family of Ras activators leading to robust elevation of Ras-GTP and engagement of the Raf-Mek-Erk kinase cascade. In response to I3A, recombinant proteins consisting of GFP fused separately to full-length RasGRP1 and RasGRP3 were rapidly recruited to cell membranes, consistent with direct binding of the compound to RasGRP's C1 domain. In the case of RasGRP3, IA3 treatment led to positive regulatory phosphorylation on T133 and activation of the candidate regulatory kinase PKCδ. I3A treatment of select B non-Hodgkin's lymphoma cell lines resulted in quantitative and qualitative changes in Bcl-2 family member proteins and induction of apoptosis, as previously demonstrated with the DAG analogue bryostatin 1 and its synthetic analogue pico. Our results offer further insights into the anticancer properties of I3A, support the idea that RasGRPs represent potential cancer therapeutic targets along with PKC, and expand the known range of ligands for RasGRP regulation

PDGFRα Regulates Follicular Cell Differentiation Driving Treatment Resistance and Disease Recurrence in Papillary Thyroid Cancer

Dedifferentiation of follicular cells is a central event in resistance to radioactive iodine and patient mortality in papillary thyroid carcinoma (PTC). We reveal that platelet derived growth factor receptor alpha (PDGFRα) specifically drives dedifferentiation in PTC by disrupting the transcriptional activity of thyroid transcription factor-1 (TTF1). PDGFRα activation dephosphorylates TTF1 consequently shifting the localization of this transcription factor from the nucleus to the cytoplasm. TTF1 is required for follicular cell development and disrupting its function abrogates thyroglobulin production and sodium iodide transport. PDGFRα also promotes a more invasive and migratory cell phenotype with a dramatic increase in xenograft tumor formation. In patient tumors we confirm that nuclear TTF1 expression is inversely proportional to PDGFRα levels. Patients exhibiting PDGFRα at time of diagnosis are three times more likely to exhibit nodal metastases and are 18 times more likely to recur within 5 years than those patients lacking PDGFRα expression. Moreover, high levels of PDGFRα and low levels of nuclear TTF1 predict resistance to radioactive iodine therapy. We demonstrate in SCID xenografts that focused PDGFRα blockade restores iodide transport and decreases tumor burden by >50%. Focused PDGFRα inhibitors, combined with radioactive iodine, represent an additional avenue for treating patients with aggressive variants of PTC

Comparison of binding affinities for ingenol 3-angelate (I3A) and phorbol 12,13-dibutyrate (PDBu).

*<p>Value from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072331#pone.0072331-Kedei1" target="_blank">[9]</a>.</p>†<p>Values represent the mean ± SEM of four independent experiments.</p>‡<p>Value represents the mean ± SEM of three independent experiments.</p

Comparison of the potencies of I3A and PMA for biochemical responses at the level of induced protein phosphorylation.

<p>A. Ramos cells were treated for 30 minutes with the indicated concentrations of I3A or PMA followed by analysis of cell lysates by immunoblotting. DMSO indicates the vehicle control. Bars indicate quantitation (mean ± SEM) of the results from three independent experiments. A representative immunoblot is illustrated. It should be noted that although the RasGRP3 antibody is not directed against a RasGRP3 phosphorylation site, it consistently yields some increase in signal under conditions of RasGRP3 phosphorylation. B. Ramos cells were treated with 3 nM I3A or PMA, in some cases after pretreatment with Gö6983 (5 µM for 40 min), and analyzed as above. Bars indicate quantitation (mean ± SEM) of the results from two independent experiments. A representative immunoblot is illustrated. An additional experiment performed under similar conditions gave similar results. C. Jurkat T cells were stimulated in a single experiment with I3A for 10 min with or without 10 min pre-incubation with the pan-PKC inhibitor bisindolymaleimide I (4.6 µM), followed by analysis of Ras-GTP, total Ras, pErk1/2 and total Erk1/2 levels.</p

Activation of RasGRP1 and RasGRP3 by I3A.

<p>A. Cultures of Rat2 cells engineered with either empty vector or RasGRP1 cDNA-expressing vector were treated with I3A or PMA for 10 minutes and compared to control cultures using the Ras-GTP pull-down assay. Results are representative of three independent experiments. B. In a single experiment Rat2 cells expressing RasGRP3 cDNA were similarly analyzed. C. Jurkat T cells were analyzed for DAG analogue-induced Ras activation in three independent experiments. D. An equal number of C57Bl/6J (WT, wildtype) and <i>Rasgrp1</i>−/− (KO, knockout) thymocytes were treated with DMSO (negative control), I3A or PMA as indicated for 10 minutes and protein lysates were compared using the phospho-Erk signaling assay. The results are representative of duplicate experiments. Quantitation of the ratios of RasGTP/Ras or p-Erk1/2/Erk1/2 are presented below the individual panels.</p

Effects of I3A on apoptosis and viable cell accumulation in B-NHL cell lines.

<p>TMRE, the percent of cells incapable of assimilating TMRE is shown.</p><p>CaspACE, the percent of cells showing activated caspases is shown.</p><p>TUNEL, the percent of cells showing nuclear DNA fragmentation is shown.</p><p>MTT, the concentration of I3A that caused 50% inhibition of viable cell accumulation was calculated by linear regression.</p>#<p>for TMRE and CaspACE assays cells were treated with 100 nM I3A for 4 days, except RL and Toledo were treated for 24 hr.</p><p>&, for TUNEL assay cells were treated with 100 nM I3A for 7 days, except RL and Toledo were treated for 2 days.</p>*<p>average of two experiments.</p>**<p>average of three experiments.</p>$<p>Rec1 cells do not stain with TMRE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072331#pone.0072331-Stang1" target="_blank">[25]</a>.</p

Quantitative and qualitative changes in Bcl-2 family members induced by I3A in representative B-NHL cells.

<p>The indicated cell lines were untreated or treated for 3 days (BL2, UPN1, Z138) or for 24 hours (RL and Toledo). Protein lysates were analyzed by immunoblotting with antibodies to the indicated proteins. Panels A and B were derived from duplicate gels. Erk was used as a loading control.</p