Non-small cell lung carcinoma cell motility, rac activation and metastatic dissemination are mediated by protein kinase C epsilon

Abstract

Background: Protein kinase C (PKC) ε, a key signaling transducer implicated in mitogenesis, survival, and cancer progression, is overexpressed in human primary non-small cell lung cancer (NSCLC). The role of PKCε in lung cancer metastasis has not yet been established.

Principal findings: Here we show that RNAi-mediated knockdown of PKCε in H358, H1299, H322, and A549 NSCLC impairs activation of the small GTPase Rac1 in response to phorbol 12-myristate 13-acetate (PMA), serum, or epidermal growth factor (EGF). PKCε depletion markedly impaired the ability of NSCLC cells to form membrane ruffles and migrate. Similar results were observed by pharmacological inhibition of PKCε with εV1-2, a specific PKCε inhibitor. PKCε was also required for invasiveness of NSCLC cells and modulated the secretion of extracellular matrix proteases and protease inhibitors. Finally, we found that PKCε-depleted NSCLC cells fail to disseminate to lungs in a mouse model of metastasis.

Conclusions: Our results implicate PKCε as a key mediator of Rac signaling and motility of lung cancer cells, highlighting its potential as a therapeutic target.

Figure 1. PMA activates Rac responses in NSCLC cells.

A. NSCLC cells were serum starved for 24 h and treated with PMA (100 nM) for different times, and Rac-GTP assays determined using a pull-down assay. A representative experiment is shown. The fold-induction in Rac-GTP levels normalized to total Rac at time = 0, as determined by densitometry, is represented in the bottom graphs. Data are expressed as mean ± S.D. (n = 3). B. Effect of the pan-PKC inhibitor GF109203X (5 µM, 30 min) on Rac activation by PMA (100 nM, 2 min).The fold-induction in Rac-GTP levels normalized to total Rac at time = 0, as determined by densitometry, is represented in the bottom graphs. Data are expressed as mean ± S.D. (n = 3). *, p<0.05; ***, p<0.001. C. A549 cells were treated with PMA (100 nM, 30 min), fixed, and stained with phalloidin-rhodamine. Top, representative micrographs are shown (n = 3). Bottom, quantification of cells bearing ruffles, expressed as mean ± S.D. of 3 individual experiments. ***, p<0.001. D.Confluent monolayers of cells were treated with PMA (100 nM, 30 min) and scraped. Closure of wounds was recorded at 9 h (A549 cells) or 20 h (H358 cells) post-treatment. Experiments were carried out in triplicate plates. Results are expressed as percentage of the closure induced by PMA, and expressed as mean ± S.E.M. (n = 3). ***, p<0.001.

Figure 2. PKCε mediates Rac activation and Rac-mediated responses induced by PMA.

A. NSCLC cells were transfected with either PKCε RNAi (ε1 or ε2) or control (CTRL) RNAi and serum starved for 24 h. A representative Western blot for PKCε expression at the time of PMA treatment is shown (n = 3). B. Cells subject to PKCε or control RNAi were stimulated with PMA (100 nM, 2 min) and Rac-GTP levels determined using a pull-down assay.A representative experiment is shown. The fold-induction in Rac-GTP levels normalized to total Rac at time = 0, as determined by densitometry, is represented in the bottom graphs. Data are expressed as mean ± S.D. (n = 3). *, p<0.05; ***, p<0.001. C. A549 cells were treated with PMA (100 nM, 30 min), fixed, and stained with phalloidin-rhodamine. Representative micrographs are shown (n = 3). D. Quantification of A549 cells bearing ruffles, expressed as mean ± S.D. of 3 individual experiments. **, p<0.01; ***, p<0.001. E. Closure of wounds in response to PMA (100 nM, 30 min) was recorded at 9 h (A549 cells). Experiments were carried out in triplicate plates. Results are expressed as percentage of the closure induced by PMA, and expressed as mean ± S.E.M. (n = 3). ***, p<0.001.

Figure 3. Rac responses induced by serum are mediated by PKCε.

A. NSCLC cells were serum starved for 24 h and then incubated with 10% FBS (5 min). Rac-GTP levels determined using a pull-down assay. A representative experiment is shown and the induction of Rac, determined by densitometry, is provided at the bottom of Western blot. Data are expressed as mean ± S.D. (n = 3). B and C. A549 cells were infected with AdVs for either β2-chimaerin (β2-chn) (Panel C) or N17-Rac1 (Panel D) at the MOIs indicated in the figure. A LacZ AdV was used as control. Migration in response to FBS was assayed using a Boyden chamber for 16 h. Left panels, representative micrographs. Right panels, quantification of 3 independent experiments. Data are expressed mean ± S.E.M. (n = 3). **, p<0.01; ***, p<0.001. D. Rac activation by 10% FBS (2 min) in A549 cells subject to either PKCε or control RNAi, as determined using a pull-down assay. A representative experiment is shown and the induction of Rac, determined by densitometry, is provided at the bottom of each Western blot. Data are expressed as mean ± S.D. (n = 3). E. Migration of A549 and H358 cellssubject to either PKCε or control RNAi in response to 10% FBS (16 h) using a Boyden chamber. Left panels, representative micrographs. Right panels, quantitation of 3 independent experiments. Data are expressed mean ± S.E.M. (n = 3). **, p<0.01; ***, p<0.001.

Figure 4. EGF-induced activation of Rac and Rac-mediated responses in A549 cells is mediated by PLC and PKCε.

A. Effect of AG1478 (0.1–1 µM) and PRI-4 (0.1–1 µM) on the activation of Rac by 10% FBS (2 min). A representative experiment is shown. Values indicate the fold-induction in Rac-GTP levels normalized to total Rac relative to levels at time = 0, as determined by densitometry. Data are expressed as mean ± S.D. (n = 3). B. Effect of EGFR depletion on the activation of Rac by 10% FBS (2 min).Values indicate the fold-induction in Rac-GTP levels normalized to total Rac at time = 0, as determined by densitometry. Data are expressed as mean ± S.D. (n = 3). C. Effect of AG1478 (1 µM) or EGFR RNAi on 10% FBS-induced A549 cell migration using a Boyden chamber (16 h). Left panels, representative micrographs. Right panel, quantification of 3 independent experiments. Data are expressed mean ± S.E.M. (n = 3). ***, p<0.001. D.A549 cells subject to either PKCε or control (CTRL) RNAi were serum starved for 24 h and stimulated with EGF (100 ng/ml, 2 min). Rac activation was determined with a pull-down assay. Data are expressed as mean ± S.D. (n = 3). E. Induction of ruffle formation by EGF (100 ng/ml, 15 min) in A549 cells subject to either PKCε or control (CTRL) RNAi. Left, representative micrographs are shown (n = 3). Right, quantification of cells bearing ruffles, expressed as mean ± S.D. of 3 individual experiments. ***, p<0.001. F. Effect of U73343 or U73122 (10 µM, 30 min) on Rac activation by either EGF (100 ng/ml) or PMA (100 nM, 2 min) in A549 cells. Values indicate the fold-induction in Rac-GTP levels normalized to total Rac at time = 0, as determined by densitometry. Data are expressed as mean ± S.D. (n = 3).

Figure 5.The. PKCε inhibitor εV1-2 impairs the formation of ruffles and migration in NSCLC cells.

A. A549 or H358 cells were serum starved for 24 h and migration in response to 10% FBS for 16 h determined using a Boyden chamber in the presence of either εV1-2 or control TAT (1 µM). Left panels, representative micrographs. Right panels, quantification of migratory cells. Data are expressed as the mean ± S.D. of 3 individual experiments. ***, p<0.001. B. Induction of ruffle formation by 10% FBS (30 min) in A549 cells in the presence of increasing concentrations of εV1-2. Left, representative micrographs. Right, quantification of cells bearing ruffles expressed as mean ± S.D. of 3 individual experiments.***, p<0.001. C. Effect of εV1-2 on Rac activation by 2% FBS (2 min) (left panel) or EGF (10 ng/ml, 2 min) (right panel). The concentration of εV1-2 in the EGF experiments was 1 µM. Representative experiments are shown. Values indicate the fold-induction in Rac-GTP levels normalized to total Rac relative to levels at time = 0, as determined by densitometry. Data are expressed as mean ± S.D. (n = 3).**, p<0.01; ***, p<0.001.

Figure 6. PKCε modulates invasion and ECM-genes in A549 cells.

A. A549 cells were transfected with RNAi for either PKCε (ε1 and ε2) or control (CTRL) and 2 days later serum-starved for additional 24 h. Cells were then seeded in Boyden chambers with Matrigel-coated membranes. Invasion was quantified 24 h later. Left, representative micrographs. Right, quantification of invasive cells. Data are represented as mean ± S.E.M. (n = 3). ***, p<0.001. B. Adhesion to Matrigel-coated plates was determined 24 h after seeding. Data are represented as mean ± S.E.M. (n = 3). C. Analysis of proteases and inhibitors of proteases by qPCR in control or PKCε-depleted cells. Results are expressed as mean ± S.E.M. (n = 4). D. Detection of ECM proteases secreted to conditioned medium (CM) from control or PKCε-depleted A549 cells. Quantification of MMP levels by densitometry is represented as mean ± S.E.M. (n = 3). **, p<0.01; ***, p<0.001. N.D., not-determined.

Figure 7. PKCε plays a role in invasion and metastatic dissemination of NSCLC cells.

A549 cells were subject to stable PKCε depletion using two different shRNA lentiviruses (ε1 and ε2) or control cells (CTRL, non-target shRNA lentivirus) and selected with puromycin. Cells were inoculated i.v. into athymic mice. A. Levels of PKCε depletion achieved in stable cells lines. A representative Western blot is shown. B. Incidence of metastasis per lung in mice sacrificed 100 days post-inoculation of cells. *, p<0.05; **,p<0.01. C. Area of tumors as determined in microscopic images. **, p<0.01. D. Representative images of lungs from mice inoculated with PKCε-depleted or control cells. Arrows, metastatic foci in whole-fixed lungs (top panels) or in lungs stained with hematoxilin and eosin (H&E) (bottom panels). E. Overall survival of mice inoculated with PKCε-depleted or control A549 cells.