A yeast-based genomic strategy highlights the cell protein networks altered by FTase inhibitor peptidomimetics

Abstract

Background: Farnesyltransferase inhibitors (FTIs) are anticancer agents developed to inhibit Ras oncoprotein activities. FTIs of different chemical structure act via a conserved mechanism in eukaryotic cells. They have low toxicity and are active on a wide range of tumors in cellular and animal models, independently of the Ras activation state. Their ultimate mechanism of action, however, remains undetermined. FTase has hundred of substrates in human cells, many of which play a pivotal role in either tumorigenesis or in pro-survival pathways. This lack of knowledge probably accounts for the failure of FTIs at clinical stage III for most of the malignancies treated, with the notable exception of haematological malignancies. Understanding which cellular pathways are the ultimate targets of FTIs in different tumor types and the basis of FTI resistance is required to improve the efficacy of FTIs in cancer treatment.

Results: Here we used a yeast-based cellular assay to define the transcriptional changes consequent to FTI peptidomimetic administration in conditions that do not substantially change Ras membrane/cytosol distribution. Yeast and cancer cell lines were used to validate the results of the network analysis. The transcriptome of yeast cells treated with FTase inhibitor I was compared with that of untreated cells and with an isogenic strain genetically inhibited for FTase activity (Deltaram1). Cells treated with GGTI-298 were analyzed in a parallel study to validate the specificity of the FTI response. Network analysis, based on gene ontology criteria, identified a cell cycle gene cluster up-regulated by FTI treatment that has the Aurora A kinase IPL1 and the checkpoint protein MAD2 as hubs. Moreover, TORC1-S6K-downstream effectors were found to be down-regulated in yeast and mammalian FTI-treated cells. Notably only FTIs, but not genetic inhibition of FTase, elicited up-regulation of ABC/transporters.

Conclusions: This work provides a view of how FTIs globally affect cell activity. It suggests that the chromosome segregation machinery and Aurora A association with the kinetochore as well as TORC1-S6K downstream effectors are among the ultimate targets affected by the transcriptional deregulation caused by FTI peptidomimetics. Moreover, it stresses the importance of monitoring the MDR response in patients treated with FTIs.

Figure 1

Effect of FTase inhibitor I on growth and Ras2 localization in wt and Δram1 cells. A, BY4741 but not Δram1 cells are sensitive to 10 μM FTase inhibitor I. Growth curves were performed in parallel cultures with or without 10 μM FTase inhibitor I. Optical density (OD₆₀₀) measurements were taken at the indicated time points. B, GFP-Ras2 localization in FTI-treated BY4741 and Δram1 cells. Total cell extracts were clarified at 3500 × g and fractionated into cytosolic (S15400 × g) and membrane (P15400 × g) fractions as previously described [50]. 30 μg of proteins were separated by SDS-PAGE and immunoblot analysis was performed using α-GFP (upper panels) after Ponceau S staining (lower panels) of nitrocellulose membranes. C, Fluorescence (upper panels) and DIC (lower panels) images of cells used in (B) prior to fractionation.

Figure 2

Comparative gene ontology analysis. Pie representation of up-regulated genes using the gene ontology GO-Slim tool at the Saccharomyces Gene Ontology Data base binned for (A) compartment of action and (B) biological processes. FTI, BY4741 cells treated with 10 μM FTase inhibitor I; GGTI, BY4741 cells treated with 10 μM GGTI298; Δram1, untreated YDL090C cells compared to untreated BY4741 cells.

Figure 3

Network analysis of the cell cycle up-regulated transcripts in FTI-treated BY4741 and in Δram1 cells. Confidence view image of gene clusters acting in the cell cycle and up-regulated in (A) 10 μM FTI treated BY4741 cells and (B) Δram1 cells using the STRING analysis tool. Analyses were performed by setting the confidence score at the highest confidence (score = 0.900) and excluding text mining. Asterisks with a red arrow indicate the input proteins. The position of nodes CDC28, MAD2, IPL1 and RPB1 within the network is highlighted.

Figure 4

Multidrug transporter GFP-Pdr5 recycling is affected by FTI treatment of wt cells. A, fluorescence (upper panels) and DIC (lower panels) images of K699 cells expressing Alr1-GFP or Gap1-GFP proteins. B, Representative fluorescence images of cells expressing PDR5-GFP with plasma membrane (PM) or endosomal (END) localization are shown in the right panels. Images were taken in a blind fashion at time 0 h (T0) or 2 h (T2 h) after 10 μM FTase inhibitor addition and at least 100 cells were counted per sample. The graph on the left represents the increase (%) of END-type over PM-type cells observed in FTI-treated versus untreated cells. The values are the mean of 3 independent experiments. Bar indicates standard deviation. 10 μM FTase inhibitor I (+FTI); untreated (NT).

Figure 5

FTI-277 treatment reduces the phosphorylation of ribosomal protein S6 in HeLa and MCF-7 cells. The levels of RPS6 (p70) phosphorylation in HeLa and MCF-7 cells were measured using the ScanR analysis software based on the intensity of AlexaFluor 555-conjugated PhoS6 antibody (Ser 235/236: Cell Signalling) recorded from images acquired from samples treated with 5 μM FTI-277 (panel FTI-277) or not treated (panel NT) in the presence of 0.1% serum for 24 h. Five wells per condition were considered. Cells were seeded in a 96-well plate and fixed and stained as described in Methods. Sixteen images were acquired randomly per well with a 20× objective using the RFP filter. The mean intensity within the red channel was automatically calculated by ScanR analysis software considering at least 600 cells. A, Images representative of FTI-277-treated and untreated HeLa cells stained with PhoS6 antibody and Hoechst, single and merged pictures are shown. B, Images representative of FTI-277-treated and untreated (NT) MCF-7 cells, stained as in (A); C, Analysis of the distribution of the phosphorylated RPS6 population in HeLa and MCF-7 cell lines. Hoechst staining was used for cell identification (main mask), as described in Methods. The minimal threshold of the PhoS6 signal was set at mean intensity 43.8 for HeLa and at 63.8 for MCF-7 cells based on the background intensity. The percentage of cells having a PhoS6 signal localised within this gate was calculated and plotted in the graph.

Figure 6

FTI treatment impairs the localization of Aurora A in G2-M cells. HeLa cells were seeded in 10 wells of a 96-well plate and grown as indicated in Methods. After 48 hours treatment with 2 μM FTI-277 (FTI-277) or no treatment (NT), the cells were fixed and stained with an antibody recognising aurora A (αIAK1; BD Biosciences) and the cell cycle distribution was calculated based on the total intensity signal in the Hoechst channel within the nuclear area using the ScanR analysis software. Ten wells were considered per sample and 12 images were acquired randomly in each well. A, Representative cells falling within the G2/M gate of treated (FTI-277) or untreated (NT) HeLa cells. B, The graph shows the percentage of G2/M phase cells that have Aurora A localized in spots at centrosomes in FTI-277-treated cells and in untreated cells. Two hundred randomly chosen cells were counted for each condition.

Figure 7

Nuclear morphology in HeLa G1 cells treated with FTI-277. The nuclear morphology of HeLa cells, grown and treated as described in Methods, was analysed throughout the cell cycle by plotting the mean intensity signal in the Hoechst channel (x) over the nuclear area (y) using the ScanR analysis software. G1 cells with an altered nuclear morphology were identified in FTI-treated samples and gated to define the mean intensity and area coordinate of this population. A gallery of images of this population is shown in (A). The same region of untreated (NT) cells is shown in (B). C, the fold change of G1 cells with an altered nuclear morphology in FTI-277 treated versus NT cells present in the gated population was calculated and plotted in the graph; the mean and the standard deviation of two independent experiments are given. D, the upper panel shows the software mask that identifies the nuclei in the images. The lower panel shows galleries of nuclei in FTI-277-treated cells present in the gated population, stained with Hoechst to label nuclei and with anti-Pak (αPaK) and anti-Phospho-histone H3 (αPhoH3) antibody as indicated