Targeting the DNA replication checkpoint by pharmacologic inhibition of Chk1 kinase : a strategy to sensitize APC mutant colon cancer cells to 5-fluorouracil chemotherapy

5-fluorouracil (5-FU) is the first line component used in colorectal cancer (CRC) therapy however even in combination with other chemotherapeutic drugs recurrence is common. Mutations of the adenomatous polyposis coli (APC) gene are considered as the initiating step of transformation in familial and sporadic CRCs. We have previously shown that APC regulates the cellular response to DNA replication stress and recently hypothesized that APC mutations might therefore influence 5-FU resistance. To test this, we compared CRC cell lines and show that those expressing truncated APC exhibit a limited response to 5-FU and arrest in G1/S-phase without undergoing lethal damage, unlike cells expressing wild-type APC. In SW480 APC-mutant CRC cells, 5-FU-dependent apoptosis was restored after transient expression of full length APC, indicating a direct link between APC and drug response. Furthermore, we could increase sensitivity of APC truncated cells to 5-FU by inactivating the Chk1 kinase using drug treatment or siRNA-mediated knockdown. Our findings identify mutant APC as a potential tumor biomarker of resistance to 5-FU, and importantly we show that APC-mutant CRC cells can be made more sensitive to 5-FU by use of Chk1 inhibitors.12 page(s

Tankyrase inhibitors stimulate the ability of tankyrases to bind axin and drive assembly of β-catenin degradation-competent axin puncta

Activation of the wnt signaling pathway is a major cause of colon cancer development. Tankyrase inhibitors (TNKSi) have recently been developed to block the wnt pathway by increasing axin levels to promote degradation of the wnt-regulator β-catenin. TNKSi bind to the PARP (poly(ADP)ribose polymerase) catalytic region of tankyrases (TNKS), preventing the PARylation of TNKS and axin that normally control axin levels through ubiquitination and degradation. TNKSi treatment of APC-mutant SW480 colorectal cancer cells can induce axin puncta which act as sites for assembly of β-catenin degradation complexes, however this process is poorly understood. Using this model system, we found that siRNA knockdown of TNKSs 1 and 2 actually blocked the ability of TNKSi drugs to induce axin puncta, revealing that puncta formation requires both the expression and the inactivation of TNKS. Immunoprecipitation assays showed that treatment of cells with TNKSi caused a strong increase in the formation of axin-TNKS complexes, correlating with an increase in insoluble or aggregated forms of TNKS/axin. The efficacy of TNKSi was antagonized by proteasome inhibitors, which stabilized the PARylated form of TNKS1 and reduced TNKSimediated assembly of axin-TNKS complexes and puncta. We hypothesise that TNKSi act to stimulate TNKS oligomerization and assembly of the TNKS-axin scaffold that form puncta. These new insights may help in optimising the future application of TNKSi in anticancer drug design.19 page(s

Rac1 augments Wnt signaling by stimulating β-catenin-lymphoid enhancer factor-1 complex assembly independent of β-catenin nuclear import

β-Catenin transduces the Wnt signaling pathway and its nuclear accumulation leads to gene transactivation and cancer. Rac1 GTPase is known to stimulate β-catenin-dependent transcription of Wnt target genes and we confirmed this activity. Here we tested the recent hypothesis that Rac1 augments Wnt signaling by enhancing β-catenin nuclear import; however, we found that silencing/inhibition or up-regulation of Rac1 had no influence on nuclear accumulation of β-catenin. To better define the role of Rac1, we employed proximity ligation assays (PLA) and discovered that a significant pool of Rac1- β-catenin protein complexes redistribute from the plasma membrane to the nucleus upon Wnt or Rac1 activation. More importantly, active Rac1 was shown to stimulate the formation of nuclear β-catenin- lymphoid enhancer factor 1 (LEF-1) complexes. This regulation required Rac1-dependent phosphorylation of β-catenin at specific serines, which when mutated (S191A and S605A) reduced β-catenin binding to LEF-1 by up to 50%, as revealed by PLA and immunoprecipitation experiments. We propose that Rac1-mediated phosphorylation of β-catenin stimulates Wnt-dependent gene transactivation by enhancing β-catenin-LEF-1 complex assembly, providing new insight into the mechanism of cross-talk between Rac1 and canonical Wnt/β-catenin signaling.14 page(s

Tankyrase inhibitors promote inclusion of axin and TNKS into insoluble complexes.

<p><b>A.</b> NIH 3T3 cells were left untreated (- CSK) or washed for 5 min with 0.2% Triton X-100 containing MT-buffer (+CSK) to permeabilize the plasma membrane and remove soluble proteins, before immunolabeling with fluorescent antibodies for axin and tubulin. Axin puncta in untreated cells and after induction by TNKSi were still visible after the detergent extraction, despite a decrease in background staining. This implies that the axin puncta are part of an insoluble pool resistant to extraction. <b>B.</b> To determine if axin and TNKS accrued in an insoluble fraction <i>in vitro</i> after TNKSi treatment in SW480 cells, cells were extracted either with RIPA buffer (left panel) or enriched for the insoluble fraction by extracting cells on the plate with SDS containing sample buffer (right panel). The addition of TNKSi (G007-LK) increased the accumulation of the TNKS-1 and -2 in the insoluble fractions and this was at least partly reduced for TNKS2 and axin following MG132 treatment, indicating that TNKSi might reduce solubility and mobility of axin and TNKS. Quantification of band intensity was measured and values were corrected to the endogenous control β-actin (a second experiment is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150484#pone.0150484.s006" target="_blank">S6 Fig</a>).</p

Model summarizing the action of TNKSi on formation of TNKS-axin complex formation and puncta assembly.

<p><b>A.</b> Table with diagrams. <b>B.</b> Hypothetical model summarizing the effects of TNKSi on TNKS/axin binding, oligomerization and puncta formation by blocking PARylation of TNKS1. This model is strengthened by a consistent negative impact of MG132 on each of these different steps including the ability of TNKSi to dePARylate TNKS, induce axin-TNKS binding complexes and to induce cytoplasmic axin-TNKS puncta. The mechanism by which MG132, through proteasome inactivation, is able to block puncta formation occurs at least in part through stabilizing the PARylation of TNKS (and possibly axin), but is yet to be fully defined. We propose the following: treatment with MG132 alone stabilizes PARylated TNKS, which causes it to dissociate from axin leading to increased nuclear axin. Treatment with TNKSi alone can stabililize unPARylated TNKS which then binds axin and retains it in the cytoplasm in large complexes and puncta. When MG132 is added after TNKSi treatment, it will induce some increase in the pool of PARylated TNKS, and causes a perinuclear shift in the TNKS/axin puncta through a mechanism yet to be defined.</p

Proteasome inhibitor induces PARylation of TNKSs and TNKSi reduces basal PARylation levels.

<p><b>A.</b> SW480 and HEK293 cells were exposed to combinations of TNKSi (G007-LK) and MG132 treatments similar to that shown in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150484#pone.0150484.g003" target="_blank">Fig 3</a>. The cells were then lysed and extracts analysed by IP assay using a specific antibody against PAR. PARylated forms of TNKS1/2 were then detected by western blot. The IgG control was negative. Pull-down of PARylated proteins revealed an increase in PARylation of TNKS1 by MG132 treatment, and a decrease of PARylation induced by TNKSi. When combined, the MG132 dominated and caused some induction of PARylated TNKS1 in both SW480 and HEK293 cells. Levels of PARylated TNKS2 (and also axin, not shown) were too low for detection. Quantification of band intensities (mean ± SD) was from two separate experiments. <b>B.</b> Total extract western blot shows similar TNKS levels after different treatments.</p

TNKSi induces the binding of TNKSs to axin.

<p><b>A.</b> SW480 cells were untreated or treated for 6 h or 24 h with 5 μM G007-LK (+/- 6 h with 20 μM MG132), then cell extracts were harvested and subjected to immunoprecipitation (IP) analysis with an axin antibody to detect axin-protein complexes. The immunoprecipitates were separated by SDS-PAGE and analysed by western blot. As shown, an increase in binding of both TNKS-1 and -2 to axin was observed after TNKSi treatments. In addition, an increase in β-catenin binding to axin was seen after 6 h treatment. In the presence of MG132, the induction of TNKS-axin complexes by G007-LK was blocked as shown. Quantification of axin-bound TNKS-1, TNKS-2 and β-catenin band intensities from two separate experiments are shown (mean ± SD). Values are corrected towards the total axin pulled down per sample. <b>B.</b> Western blot of total protein extract demonstrates that total levels of TNKSs were not modified by TNKSi treatment, but displayed a modest increase after 6 h MG132. The G007-LK treatment did modestly increase axin levels (and decrease β-catenin levels after 24 h treatment) as expected. This control blot indicates that the strong induction of TNKS-axin complexes above is not due to changes in protein expression. <b>C.</b> A diagram outlining how these novel effects of TNKSi on axin-TNKS complexes may contribute to axin/TNKS positive puncta formation in the cell.</p

TNKSi treatment can induce axin expression and axin puncta associated with β-catenin degradation.

<p><b>A.</b> Western blot analysis of different cell lines treated +/- for 24 h with 2.5 μM of the TNKSi XAV939 revealed an increase in axin expression in different cell lines, and rescue of the degradation of β-catenin in SW480 CRC cells where the wnt pathway is disrupted by APC mutation. β-actin levels are shown as loading controls. Quantification of band intensities from two separate blots was performed and normalized to actin. The values shown are mean ± SD. <b>B.</b> SW480 cells were treated with 5 μM of the TNKSi G007-LK for 24 h, then immunostained with specific antibodies to detect different components of the β-catenin degradation complex located at axin puncta. The following combinations of proteins were co-stained: axin and total β-catenin, APC and Axin, GSK3β and Axin and phosphorylated and non-phosphorylated β-catenin. Cells were imaged using a DeltaVision microscope system. <b>C.</b> SW480 control cells or cells treated with 5 μM of the TNKSi G007-LK for 24 h were stained for fluorescence. In control cells a disperse staining pattern is observed whereas in TNKSi treated cells the TNKS-1 and -2 (red) specifically colocalize at TNKSi induced axin puncta (green) as shown by immunofluorescence microscopy.</p

Induction of axin puncta by TNKSi is proteasome-dependent.

<p><b>A.</b> SW480 cells were treated with single or combined doses of tankyrase (5 μM G007LK) and proteasome (20 μM MG132) inhibitors. The MG132 was added for 6 h, either simultaneously with G007-LK for a 6 h treatment, or during the last 6 h of a 24 h G007-LK treatment. As shown in the immunofluorescence images, the addition of MG132 caused a reduction in the % of cells with visible TNKSi-induced axin puncta after 6 h treatment. The 6 h MG132 treatment also caused a modest increase in nuclear staining of axin. The later addition of MG132 (at the end of a 24 h G007-LK treatment) caused the induced axin puncta to relocate to the perinuclear region. <b>B.</b> The number of axin puncta per cell were scored by microscopy and categorized (<5, 5–15 or >15 puncta per cell). As shown, TNKSi induced a high number of puncta per cell (more than 80% of cells scored >15 puncta per cell) and this was blocked at 6 h or reduced after 24 h by MG132 treatment. <b>C.</b> A diagram summarizing the effect of G007-LK +/- MG132 on axin pattern and cellular distribution (shown in green).</p

Stroma-derived but not tumor ADAMTS1 is a main driver of tumor growth and metastasis

The matrix metalloprotease ADAMTS1 (A Disintegrin And Metalloprotease with ThromboSpondin repeats 1) has been involved in tumorigenesis although its contributions appeared ambiguous. To understand the multifaceted actions of this protease, it is still required a deeper knowledge of its implication in heterogeneous tumor-stroma interactions. Using a syngeneic B16F1 melanoma model in wild type and ADAMTS1 knockout mice we found distinct stroma versus tumor functions for this protease. Genetic deletion of ADAMTS1 in the host microenvironment resulted in a drastic decrease of tumor growth and metastasis. However, the downregulation of tumor ADAMTS1 did not uncover relevant effects. Reduced tumors in ADAMTS1 KO mice displayed a paradoxical increase in vascular density and vascular-related genes; a detailed characterization revealed an impaired vasculature, along with a minor infiltration of macrophages. In addition, ex-vivo assays supported a chief role for ADAMTS1 in vascular sprouting, and melanoma xenografts showed a relevant induction of its expression in stroma compartments. These findings provide the first genetic evidence that supports the pro-tumorigenic role of stromal ADAMTS1