Treatment of cancer cells with Lapatinib negatively regulates general translation and induces stress granules formation

Abstract

Stress granules (SG) are cytoplasmic RNA granules that form during various types of stress known to inhibit general translation, including oxidative stress, hypoxia, endoplasmic reticulum stress (ER), ionizing radiations or viral infection. Induction of these SG promotes cell survival in part through sequestration of proapoptotic molecules, resulting in the inactivation of cell death pathways. SG also form in cancer cells, but studies investigating their formation upon treatment with chemotherapeutics are very limited. Here we identified Lapatinib (Tykerb / Tyverb®), a tyrosine kinase inhibitor used for the treatment of breast cancers as a new inducer of SG in breast cancer cells. Lapatinib-induced SG formation correlates with the inhibition of general translation initiation which involves the phosphorylation of the translation initiation factor eIF2α through the kinase PERK. Disrupting PERK-SG formation by PERK depletion experiments sensitizes resistant breast cancer cells to Lapatinib. This study further supports the assumption that treatment with anticancer drugs activates the SG pathway, which may constitute an intrinsic stress response used by cancer cells to resist treatment.

Fig 1. LAP induces SG in T47D.

(A) T47D were treated with LAP (20 μM) for two hours. Cells were fixed, permeabilized and processed for immunofluorescence using antibodies against different SG markers (FMRP, G3BP, DDX3, eIF4GI). DAPI is used as a marker for nuclei. Scale bars are shown. The indicated percentage of SG-positive cells (>3 granules / cell) representing more than 1000 cells of five independent experiments. Arsenite (0.5 mM) treatment was used as a control of SG formation. (B) T47D were treated with LAP for two hours. Cells were fixed, permeabilized and processed for immunofluorescence using specific antibodies against the SG marker FMRP and m⁶A. DAPI is used as a marker for nuclei. (C) T47D were treated with LAP, collected at the indicated time points and analysed for SG formation as above. The percentage of SG was calculated as in 1A. (D) T47D were treated with LAP (20 μM) alone or with cycloheximide (100 μg / ml) for two hours, fixed, permeabilized and processed for immunofluorescence using anti-FMRP antibodies to detect SG. DAPI is used as a marker for nuclei. The percentage of SG-positive cells is indicated.

Fig 2. Formation of SG that occurs during Lap treatment correlates with the activation of PERK-PeIF2α pathway and inhibition of general translation.

(A-C) T47D were treated with LAP (20 μM) for two hours. (A) Protein extracts were prepared and their content analysed by western blot using antibodies specific to the indicated proteins. Tubulin (Tub) and eIF2α serve as loading controls. (B) Five minutes before the end of the treatment, puromycin (50 μg / ml) was added. Cells were collected and protein content was analyzed by western blot for puromycin incorporation into nascent polypeptide chains using anti-puromycin antibodies (top panel). Red ponceau staining shows equal protein loading (bottom panel). (C) Cytoplasmic extracts were prepared and fractionated onto 15–55% sucrose gradients and the polyribosomes profiles were recorded by measuring the OD₂₅₄. Positions of 40S, 60S, monosomes and polysomes are indicated.

Fig 3. PERK is required for SG formation during LAP treatment.

(A) T47D were treated with LAP (20 μM) or with LAP and ISRIB (100 nM) for two hours. Cells were processed for immunofluorescence as above using anti-FMRP and anti-FXR1 antibodies. DAPI stains nuclei. (B-D) T47D were treated with two specific PERK siRNAs and then incubated with LAP (20 μM) for two hours. (B) Cells were fixed and SG were visualised by immunofluorescence using anti-FMRP and anti-FXR1 antibodies. DAPI is used as a marker for nuclei. (C) Cells were collected, and protein content was analyzed by western blot for PERK and PeIF2α. eIF2α serves as loading control. PERK level was estimated by densitometry quantification of the corresponding signal and then normalized to eIF2α. These quantifications revealed a reproducible 70 to 75% PERK depletion. (D) Five minutes before the end of the LAP treatment (20 μM), puromycin (50 μg / ml) was added. Cells were collected and protein content was analyzed by western blot for puromycin incorporation into nascent polypeptide chains using anti-puromycin antibodies (top panel). Coomassie Blue staining shows equal protein loading (bottom panel). Puromycin signals were estimated by densitometry quantification and then normalized to total protein loading assessed by coomassie blue staining and reported as a percentage of the untreated cells.

Fig 4. PERK depletion sensitizes T47D to LAP.

(A) The indicated shRNA-expressing T47D were treated with LAP (20 μM) for twenty-four hours, washed with PBS and MTT solvent was added at the indicated time points for an additional two hours. The absorbance was monitored at 560 nm. Cell viability was calculated as the percentage of the absorbance values of LAP-treated cells relative to the absorbance values corresponding to untreated cells. Values are presented as the mean percentage +/- SD. (B) Depletion of PERK in shRNA PERK-expressing T47D was validated by western blot analysis using anti-PERK antibodies and quantified as above.