TLN-4601 suppresses growth and induces apoptosis of pancreatic carcinoma cells through inhibition of Ras-ERK MAPK signaling

Abstract Background TLN-4601 is a structurally novel farnesylated dibenzodiazepinone discovered using Thallion's proprietary DECIPHER® technology, a genomics and bioinformatics platform that predicts the chemical structures of secondary metabolites based on gene sequences obtained by scanning bacterial genomes. Our recent studies suggest that TLN-4601 inhibits the Ras-ERK MAPK pathway post Ras prenylation and prior to MEK activation. The Ras-ERK MAPK signaling pathway is a well-validated oncogenic cascade based on its central role in regulating the growth and survival of cells from a broad spectrum of human tumors. Furthermore, RAS isoforms are the most frequently mutated oncogenes, occurring in approximately 30% of all human cancers, and KRAS is the most commonly mutated RAS gene, with a greater than 90% incidence of mutation in pancreatic cancer. Results To evaluate whether TLN-4601 interferes with K-Ras signaling, we utilized human pancreatic epithelial cells and demonstrate that TLN-4601 treatment resulted in a dose- and time-dependent inhibition of Ras-ERK MAPK signaling. The compound also reduced Ras-GTP levels and induced apoptosis. Finally, treatment of MIA PaCa-2 tumor-bearing mice with TLN-4601 resulted in antitumor activity and decreased tumor Raf-1 protein levels. Conclusion These data, together with phase I/II clinical data showing tolerability of TLN-4601, support conducting a clinical trial in advanced pancreatic cancer patients

Src-Mediated Phosphorylation of the Tyrosine Phosphatase PRL-3 Is Required for PRL-3 Promotion of Rho Activation, Motility and Invasion

The metastasis-associated tyrosine phosphatase PRL-3/PTP4A is upregulated in numerous cancers, but the mechanisms modulating PRL-3 activity other than its expression levels have not been investigated. Here we report evidence for both Src-dependent tyrosine phosphorylation of PRL-3 and Src-mediated regulation of PRL-3 biological activities. We used structural mutants, pharmacological inhibitors and siRNA to demonstrate Src-dependent phosphorylation of endogenous PRL-3 in SW480 colon cancer cells. We also demonstrated that PRL-3 was not tyrosine phosphorylated in SYF mouse embryo fibroblasts deficient in Src, Yes and Fyn unless Src was re-expressed. Further, we show that platelet-derived growth factor (PDGF) can stimulate PRL-3 phosphorylation in a Src-dependent manner. Finally, we show that PRL-3-induced cell motility, Matrigel invasion and activation of the cytoskeleton-regulating small GTPase RhoC were abrogated in the presence of the phosphodeficient PRL-3 mutant Y53F, or by use of a Src inhibitor. Thus, PRL-3 requires the activity of a Src kinase, likely Src itself, to promote these cancer-associated phenotypes. Our data establish a model for the regulation of PRL-3 by Src that supports the possibility of their coordinate roles in signaling pathways promoting invasion and metastasis, and supports simultaneous use of novel molecularly targeted therapeutics directed at these proteins

High Affinity for Farnesyltransferase and Alternative Prenylation Contribute Individually to K-Ras4B Resistance to Farnesyltransferase Inhibitors

Farnesyltransferase inhibitors (FTIs) block Ras farnesylation, subcellular localization and activity, and inhibit the growth of Ras-transformed cells. Although FTIs are ineffective against K-Ras4B, the Ras isoform most commonly mutated in human cancers, they can inhibit the growth of tumors containing oncogenic K-Ras4B, implicating other farnesylated proteins or suggesting distinct functions for farnesylated and for geranylgeranylated K-Ras, which is generated when farnesyltransferase is inhibited. In addition to bypassing FTI blockade through geranylgeranylation, K-Ras4B resistance to FTIs may also result from its higher affinity for farnesyltransferase. Using chimeric Ras proteins containing all combinations of Ras background, CAAX motif, and K-Ras polybasic domain, we show that either a polybasic domain or an alternatively prenylated CAAX renders Ras prenylation, Ras-induced Elk-1 activation, and anchorage-independent cell growth FTI-resistant. The polybasic domain alone increases the affinity of Ras for farnesyltransferase, implying independent roles for each K-Ras4B sequence element in FTI resistance. Using microarray analysis and colony formation assays, we confirm that K-Ras function is independent of the identity of the prenyl group and, therefore, that FTI inhibition of K-Ras transformed cells is likely to be independent of K-Ras inhibition. Our results imply that relevant FTI targets will lack both polybasic and potentially geranylgeranylated methionine-CAAX motifs

K-Ras4A splice variant is widely expressed in cancer and uses a hybrid membrane-targeting motif

The KRAS oncogene is mutated more frequently in human cancer than any other. The KRAS transcript is alternatively spliced to give rise to two products, K-Ras4A and K-Ras4B, both of which are oncogenic when KRAS is mutated. We detected significant amounts of each transcript in human tumor cells and colorectal carcinomas. We found that K-Ras4A is targeted to the plasma membrane by dual targeting motifs distinct from those of K-Ras4B. Because interfering with membrane association of Ras proteins remains one of the most attractive approaches to anti-Ras therapy, efforts in this direction will have to disrupt both the K-Ras4A and the K-Ras4B membrane-targeting pathways

Nanoparticle formulations of histone deacetylase inhibitors for effective chemoradiotherapy in solid tumors

Histone deacetylase inhibitors (HDACIs) represent a class of promising agents that can improve radiotherapy in cancer treatment. However, the full therapeutic potential of HDACIs as radiosensitizers has been restricted by limited efficacy in solid malignancies. In this study, we report the development of nanoparticle (NP) formulations of HDACIs that overcome these limitations, illustrating their utility to improve the therapeutic ratio of the clinically established first generation HDACI vorinostat and a novel second generation HDACI quisinostat. We demonstrate that NP HDACIs are potent radiosensitizers in vitro and are more effective as radiosensitizers than small molecule HDACIs in vivo using mouse xenograft models of colorectal and prostate carcinomas. We found that NP HDACIs enhance the response of tumor cells to radiation through the prolongation of γ-H2AX foci. Our work illustrates an effective method for improving cancer radiotherapy treatment

PRL-3 is phosphoryated in vitro by the Src tyrosine kinase, and endogenous PRL-3 is tyrosine-phosphorylated in cells.

<p>A) Schematic diagram of PRL-3 showing the relative positions of the six tyrosines evaluated in these studies, as well as the C-terminal CaaX motif. The numbers shown reflect the probability of phosphorylation at each site based on predictions by NetPhos 2.0. B) PRL-3 (“WT”) or a mutant in which all six tyrosines were substituted by phenylalanine (“All_F”) was fused to GST, purified from bacteria, and subjected to <i>in vitro</i> phosphorylation with purified Src. WT PRL-3 was phosphorylated by Src while the “All_F” PRL-3 mutant was not. Src itself is visible on the phosphotyrosine blot because it becomes autophosphorylated on Y416 (pSrc). Coomassie staining demonstrates equal amounts of PRL-3 protein in each reaction. C) To determine whether endogenous PRL-3 is also tyrosine phosphorylated in cells, SW480 cells were treated with the tyrosine phosphatase inhibitor pervanadate (VO₄, 100 µM, 1 h) to enhance detection of transient tyrosine phosphorylation events. Endogenous PRL-3 was immunoprecipitated using anti-PRL-3 antibody, followed by SDS-PAGE and western blot analysis for either PRL-3 or phosphotyrosine.</p

PRL-3 requires the activity of a Src family kinase to promote invasion and RhoC activation.

<p>A) SW480 cells expressing WT PRL-3 were subjected to Matrigel invasion analysis as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064309#pone-0064309-g005" target="_blank">Figure 5</a>. The Src family kinase inhibitor SU6656 was added to both the top and bottom chambers at the indicated concentrations. Cells expressing phosphatase-inactive PRL-3 (C104S) served as a negative control. Data are shown normalized to basal levels of invasion of vector-expressing cells. Results from four assays are shown +/− SD. B) SW480 cells expressing WT PRL-3 were treated with the Src inhibitor, SU6656, and analyzed for levels of active, GTP-bound RhoC using a Rhotekin-RBD pull down assay. Images from a representative assay are shown. Lanes containing samples not relevant to this work have been removed. All panels were taken from the same assay using the same photographic exposure time. Phosphatase-inactive PRL-3 (C104S) served as a negative control. C) Data quantitated from at least three pull down assays as in panel B are shown +/− SEM.</p

PRL-3 promotion of invasion and cell motility requires an intact Y53.

<p>A) SW480 cells stably expressing WT PRL-3 (WT) or a phosphorylation-deficient mutant lacking tyrosine 53 (Y53F) were subjected to Matrigel invasion assays. Data from three independent assays are shown. For each sample, 5x10⁴ cells were placed in the top chamber in the absence of serum; the bottom chamber contained medium supplemented with 10% serum. After 72 h, invaded cells were fixed, stained and counted. Equal expression of WT HA-tagged PRL-3 and the Y53F mutant was confirmed by HA blot. B) H1299 cells were transiently transfected with EGFP-tagged PRL-3, the Y53F mutant or vector only. The motility rate of individual cells was evaluated using a BioStation IM live cell recorder. Data shown are from three independent assays, and include a total of at least 35 cells for each sample. Images of representative cells expressing each EGFP-tagged protein are shown below the graph. All images were captured under identical microscopic and photographic settings. Data in both A and B are shown +/− SEM, and <i>p</i>-values were calculated using a two-tailed Student's <i>t</i>-test.</p