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RAC1 inhibition as a therapeutic target for gefitinib-resistant non-small-cell lung cancer.

Kaneto N, Yokoyama S, Hayakawa Y, Kato S, Sakurai H, Saiki I - Cancer Sci. (2014)

Bottom Line: Although epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (EGFR-TKI), including gefitinib, provide a significant clinical benefit in non-small-cell lung cancer (NSCLC) patients, the acquisition of drug resistance has been known to limit the efficacy of EGFR-TKI therapy.In addition, these suppressions by RAC1 inhibition were mediated through MEK or PI3K independent mechanisms.Collectively, these results open up a new opportunity to control the cancer progression by targeting the RAC1 pathway to overcome the resistance to EGFR-TKI in NSCLC patients.

View Article: PubMed Central - PubMed

Affiliation: Division of Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama, Toyama, Japan.

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Constitutively active RAC1 attenuates anti-migratory effect of gefitinib. (a) PC-9 cells were co-transfected with RAC1G12V or RAC1Q61L expression vectors and EGFP expression vector. After 24 h, cells were treated with 300 nM gefitinib (gef) for 24 h and subjected to migration assay. Migrated EGFP+ cells were counted manually under a fluorescence microscope at 50× magnification. Data are the means ± SD of at least three independent experiments. *P < 0.01 by two-way anova followed by the Bonferroni post-hoc test. (b) Lamellipodia+ cells in EGFP+ cells were counted at 20× magnification after staining with rhodamine-conjugated phalloidin and DAPI. Other conditions are similar to (a). Data are the means ± SD of at least three independent experiments. *P < 0.01 by two-way anova followed by the Bonferroni post-hoc test. (c) PC-9 cells were treated similarly to (a). Whole cell lysates from the transfected PC-9 cells were subjected to western blotting.
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fig03: Constitutively active RAC1 attenuates anti-migratory effect of gefitinib. (a) PC-9 cells were co-transfected with RAC1G12V or RAC1Q61L expression vectors and EGFP expression vector. After 24 h, cells were treated with 300 nM gefitinib (gef) for 24 h and subjected to migration assay. Migrated EGFP+ cells were counted manually under a fluorescence microscope at 50× magnification. Data are the means ± SD of at least three independent experiments. *P < 0.01 by two-way anova followed by the Bonferroni post-hoc test. (b) Lamellipodia+ cells in EGFP+ cells were counted at 20× magnification after staining with rhodamine-conjugated phalloidin and DAPI. Other conditions are similar to (a). Data are the means ± SD of at least three independent experiments. *P < 0.01 by two-way anova followed by the Bonferroni post-hoc test. (c) PC-9 cells were treated similarly to (a). Whole cell lysates from the transfected PC-9 cells were subjected to western blotting.

Mentions: To further determine whether RAC1 suppression is involved in the suppression of cell migration upon gefitinib treatment, we generated PC-9 cells overexpressing constitutive active forms of RAC1, RAC1G12V or RAC1Q61L.(24) As shown in Figure 3(a), the reduced cell migration of PC-9 cells by gefitinib treatment was diminished in RAC1G12V or RAC1Q61L overexpressing PC-9 cells. Furthermore, the reduction of lamellipodia formation (Fig. 3b) by gefitinib treatment was also diminished in both RAC1G12V and RAC1Q61L overexpressing cells. RAC1 overexpression was confirmed by detection of HA tag (Fig. 3c). Collectively, these results strongly support that RAC1 activity is critically involved in EGFR-mediated cell migration of PC-9 cells.


RAC1 inhibition as a therapeutic target for gefitinib-resistant non-small-cell lung cancer.

Kaneto N, Yokoyama S, Hayakawa Y, Kato S, Sakurai H, Saiki I - Cancer Sci. (2014)

Constitutively active RAC1 attenuates anti-migratory effect of gefitinib. (a) PC-9 cells were co-transfected with RAC1G12V or RAC1Q61L expression vectors and EGFP expression vector. After 24 h, cells were treated with 300 nM gefitinib (gef) for 24 h and subjected to migration assay. Migrated EGFP+ cells were counted manually under a fluorescence microscope at 50× magnification. Data are the means ± SD of at least three independent experiments. *P < 0.01 by two-way anova followed by the Bonferroni post-hoc test. (b) Lamellipodia+ cells in EGFP+ cells were counted at 20× magnification after staining with rhodamine-conjugated phalloidin and DAPI. Other conditions are similar to (a). Data are the means ± SD of at least three independent experiments. *P < 0.01 by two-way anova followed by the Bonferroni post-hoc test. (c) PC-9 cells were treated similarly to (a). Whole cell lysates from the transfected PC-9 cells were subjected to western blotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig03: Constitutively active RAC1 attenuates anti-migratory effect of gefitinib. (a) PC-9 cells were co-transfected with RAC1G12V or RAC1Q61L expression vectors and EGFP expression vector. After 24 h, cells were treated with 300 nM gefitinib (gef) for 24 h and subjected to migration assay. Migrated EGFP+ cells were counted manually under a fluorescence microscope at 50× magnification. Data are the means ± SD of at least three independent experiments. *P < 0.01 by two-way anova followed by the Bonferroni post-hoc test. (b) Lamellipodia+ cells in EGFP+ cells were counted at 20× magnification after staining with rhodamine-conjugated phalloidin and DAPI. Other conditions are similar to (a). Data are the means ± SD of at least three independent experiments. *P < 0.01 by two-way anova followed by the Bonferroni post-hoc test. (c) PC-9 cells were treated similarly to (a). Whole cell lysates from the transfected PC-9 cells were subjected to western blotting.
Mentions: To further determine whether RAC1 suppression is involved in the suppression of cell migration upon gefitinib treatment, we generated PC-9 cells overexpressing constitutive active forms of RAC1, RAC1G12V or RAC1Q61L.(24) As shown in Figure 3(a), the reduced cell migration of PC-9 cells by gefitinib treatment was diminished in RAC1G12V or RAC1Q61L overexpressing PC-9 cells. Furthermore, the reduction of lamellipodia formation (Fig. 3b) by gefitinib treatment was also diminished in both RAC1G12V and RAC1Q61L overexpressing cells. RAC1 overexpression was confirmed by detection of HA tag (Fig. 3c). Collectively, these results strongly support that RAC1 activity is critically involved in EGFR-mediated cell migration of PC-9 cells.

Bottom Line: Although epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (EGFR-TKI), including gefitinib, provide a significant clinical benefit in non-small-cell lung cancer (NSCLC) patients, the acquisition of drug resistance has been known to limit the efficacy of EGFR-TKI therapy.In addition, these suppressions by RAC1 inhibition were mediated through MEK or PI3K independent mechanisms.Collectively, these results open up a new opportunity to control the cancer progression by targeting the RAC1 pathway to overcome the resistance to EGFR-TKI in NSCLC patients.

View Article: PubMed Central - PubMed

Affiliation: Division of Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama, Toyama, Japan.

Show MeSH
Related in: MedlinePlus