Specific Oncogenic Activity of the Src-Family Tyrosine Kinase c-Yes in Colon Carcinoma Cells

c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of β-catenin at cell membranes and a reduction of expression of β-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in β-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src

Targeting Bcl-2/Bcl-XL induces antitumor activity in uveal melanoma patient-derived xenografts.

Uveal melanoma (UM) is associated with a high risk of metastases and lack of efficient therapies. Reduced capacity for apoptosis induction by chemotherapies is one obstacle to efficient treatments. Human UM is characterized by high expression of the anti-apoptotic protein Bcl-2. Consequently, regulators of apoptosis such as Bcl-2 family inhibitors may constitute an attractive approach to UM therapeutics. In this aim, we have investigated the efficacy of the Bcl-2/Bcl-XL inhibitor S44563 on 4 UM Patient-Derived Xenografts (PDXs) and derived-cell lines.Four well characterized UM PDXs were used for in vivo experiments. S44563 was administered alone or combined with fotemustine either concomitantly or after the alkylating agent. Bcl-2, Bcl-XL, and Mcl-1 expressions after S44563 administration were evaluated by immunohistochemistry (IHC).S44563 administered alone by at 50 and 100 mg/kg i.p. induced a significant tumour growth inhibition in only one xenograft model with a clear dose effect. However, when S44563 was concomitantly administered with fotemustine, we observed a synergistic activity in 3 out of the 4 tested models. In addition, S44563 administered after fotemustine induced a tumour growth delay in 2 out of 3 tested xenografts. Finally, IHC analyses showed that Bcl-2, Bcl-XL, and Mcl-1 expression were not modified after S44563 administration.The novel anti-apoptotic experimental compound S44563, despite a relative low efficacy when administered alone, increased the efficacy of fotemustine in either concomitant or sequential combinations or indeed subsequent to fotemustine. These data support further exploration of potential therapeutic effect of Bcl-2/Bcl-xl inhibition in human UM

Immunohistochemical analyses under S44563 administration.

<p>Immunohistochemical analyses of the MM66 UM xenograft after S44563, fotemustine, or S44563+fotemustine.</p

TGI and complete remission induced by a combination of S44563 and fotemustine.

<p>Complete remissions are indicated in brackets.</p><p>p<0.005.</p

Inhibition of the interaction between Bcl-2 or Bcl-X_L and fluorescent Puma BH3 peptide measured by the decrease of fluorescence polarisation as a function of S44563-2 concentration.

<p>Three independent experiments are presented. FP data are presented in millipolarization units (mP). For each experiment, measures are assessed in triplicate.</p

<i>In vivo</i> responses of UM PDXs to S44563 administered alone or in combination with fotemustine.

<p>A. MP41 xenograft was treated either by S44563 at 50/kg (▪) or 100 mg/kg (□) 5 days a week for 5 weeks. <b>B.</b> Overall survival of mice bearing MP41 tumors treated by S44563 (O), fotemustine (▪), and concomitant fotemustine+S44563 (⧫). <b>C.</b> MP77 xenograft was treated by S44563 at 100 mg/kg (□), fotemustine 15 mg/kg (Δ), or both (▴). <b>D.</b> MM66 xenograft was treated by S44563 at 50 mg/kg (□), fotemustine 30 mg/kg (Δ), or both (▴). Mice in the control group (•) received 0,3 ml of the drug-formulating vehicle with the same schedule as the treated animals. Tumor growth was evaluated by plotting the mean of the RTV (relative tumor volume) ± SD per group. Between 8 to 12 mice per group were included in <i>in vivo</i> experiments.</p

<i>In vitro</i> apoptosis induction by S44563.

<p>A and B. Apoptosis induction by S44563. The MP41, MM26, and MM66 cell lines were incubated with 17 µM and 34 µM S44563 for 24 hours, after which the samples were labeled with DAPI/annexin V-FITC (A) or JC1 (B). The proportion of annexin V-positive cells and low Δψ_m cells was indicated in C and E, respectively. A two-way ANOVA with Bonferroni post-test was then performed (<b>*</b> p<0.05). <b>C.</b> Cell cycle analyses after S44563 exposure (17 or 34 µM for 24 h) in the 3 UM cell lines. Cell cycle analysis was determined by labeling the DNA with propidium iodide. Each of the three lines was treated by 17 µM or 34 µM of S44563. The proportion of cells (%) in different cell cycle phases was compared with the control (untreated). Two-way ANOVA with Bonferroni post-test was then performed (<b>*</b> means a p<0.05). <b>D.</b> Cell cycle analyses of the xMP41 UM cell line. I and J. Measurement of autophagy on the 3 UM cell lines after S44563 exposure (2 or 4 µM for 24 h). After S44563 treatment, the amount of active and inactive protein LC3 was determined and reported to ß-actine. The quantity of total LC3 protein was calculated to study the activation of LC3. Results were presented as percentage and total amount of active cleaved LC3, relative to β-actin. A two-way ANOVA with Bonferroni post-test was then performed (<b>*</b> means a p<0.05).</p

Overall and median survival after combination of S44563 and fotemustine.

<p><b>Abbreviations:</b> F-30/S-50, fotemustine followed by S44563 adminstration since day 43; OS (day), overall survival observed at the indicated day; Median S, median survival in days since start of treatment.</p><p>F-30 versus F-30+S-50 or F-30/S-100: p = 0.04.</p><p>F-30/S-100 versus F-30+S-50/S-100: p = 0.038.</p