Targeting PAK4 reverses cisplatin resistance in NSCLC by modulating ER stress

Abstract

Chemoresistance poses a significant impediment to effective treatments for non-small-cell lung cancer (NSCLC). P21-activated kinase 4 (PAK4) has been implicated in NSCLC progression by invasion and migration. However, the involvement of PAK4 in cisplatin resistance is not clear. Here, we presented a comprehensive investigation into the involvement of PAK4 in cisplatin resistance within NSCLC. Our study revealed enhanced PAK4 expression in both cisplatin-resistant NSCLC tumors and cell lines. Notably, PAK4 silencing led to a remarkable enhancement in the chemosensitivity of cisplatin-resistant NSCLC cells. Cisplatin evoked endoplasmic reticulum stress in NSCLC. Furthermore, inhibition of PAK4 demonstrated the potential to sensitize resistant tumor cells through modulating endoplasmic reticulum stress. Mechanistically, we unveiled that the suppression of the MEK1-GRP78 signaling pathway results in the sensitization of NSCLC cells to cisplatin after PAK4 knockdown. Our findings establish PAK4 as a promising therapeutic target for addressing chemoresistance in NSCLC, potentially opening new avenues for enhancing treatment efficacy and patient outcomes.

Fig. 1. PAK4 was upregulated in cisplatin-resistant NSCLC.

A mRNA expression of PAK4 was analyzed in 20 cisplatin-sensitive and matched cisplatin-resistant tissues. B Western blot analysis of PAK4 expression in the paired tissues. C Representative immunohistochemical staining (left panels) and statistical analysis (right panels) were shown in 55 cisplatin-sensitive NSCLC tissues and 104 cisplatin-resistant NSCLC tissues. Scale bars 100 μm. D, E Cells were treated with 5 μmol/L cisplatin for 48 h and cell viability was measured by CCK8 assay. F, G Real-time PCR and western blot were analyzed PAK4 levels. Data are shown as the means of three independent experiments or representative data. Data are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01 by Student’s t test.

Fig. 2. PAK4 knockdown enhanced NSCLC cell chemosensitivity.

A, B A549-res cells (A) and NCI-H520-res cells (B) stably expressing PAK4 shRNA (shPAK4) or control shRNA (shCtrl) were treated with the indicated doses of cisplatin for 48 h and cell viability was measured by CCK8 assay. Data are expressed as mean ± SEM. C, D A549-res cells and NCI-H520-res cells stably expressing PAK4 shRNA (shPAK4) or control shRNA (shCtrl) were treated with cisplatin (5 μmol/L) for 48 h. Colony-forming ability for A549-res cells (C). Colony-forming ability for NCI-H520-res cells (D). E Flow cytometry analyses of the percentages of apoptotic cells. F The levels of apoptosis markers (cleaved casp-3, cleaved casp-9 and cleaved PARP) were examined by western blot analysis. Data are shown as the means of three independent experiments or representative data. Data are expressed as mean ± standard deviation. **P < 0.01, ***P < 0.001 by Student’s t test.

Fig. 3. Cisplatin evoked ER stress in NSCLC cells.

A GRP78 expression was examined in A549 and NCI-H520 cells following treatment with cisplatin (5 μmol/L). Floating and adherent cells were lysed at 0, 3, 6, 9, 18, and 36 h and analyzed by western blot analysis. B GRP78 expression was examined in A549 and NCI-H520 cells following treatment with tunicamycin (200 ng/μl). Floating and adherent cells were lysed at 0, 3, 6, 9, 18, and 36 h and analyzed by western blot analysis. C, D Treated with 5 µmol/L cisplatin for 0, 9, and 36 h, A549 cells (C) and NCI-H520 cells (D) were loaded with Fluo-3am for 30 min. Cytoplasmic calcium level was examined by flow cytometry assay. Data are expressed as mean ± standard deviation. E, F Molecular markers of the endoplasmic reticulum stress signaling pathway were detected by western blot analysis in A549 and NCI-H520. Data are shown as the means of three independent experiments or representative data. Data are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01 by Student’s t test.

Fig. 4. PAK4 knockdown chemosensitized NSCLC cells via modulating ER stress.

A PAK4 and GRP78 expression profiles were examined by western blot analysis in A549/A549-res and NCI-H520/NCI-H520-res cells. B A549-res and NCI-H520-res cells stably expressing PAK4 shRNA (shPAK4) or control shRNA (shCtrl) were treated with 5 µmol/L cisplatin for 48 h. PAK4 and GRP78 expression was examined by western blot analysis (left panel) and Western blot analyses of PAK4 and GRP78 expression in PAK4 overexpressed cells (right panel). C A549-res and NCI-H520-res cells stably expressing PAK4 shRNA (shPAK4) or control shRNA (shCtrl) or shPAK4 and GRP78 plasmids were treated with 5 µmol/L cisplatin for 48 h. Cytoplasmic calcium level was examined by flow cytometry assay. D A549-res and NCI-H520-res cells stably expressing PAK4 shRNA (shPAK4) or control shRNA (shCtrl) were transfected with GRP78 or control vector, followed by treatment with 5 µmol/L cisplatin for 48 h. Apoptosis rates were determined by flow cytometry. Data are shown as the means of three independent experiments or representative data. Data are expressed as mean ± standard deviation. **P < 0.01, ***P < 0.001 by Student’s t test.

Fig. 5. PAK4 knockdown chemosensitized cisplatin-resistant NSCLC xenografts in vivo.

A Scheme indicating the timing of xenografting and longitudinal treatment. B A549-res cells stably expressing shPAK4 and luciferase reporter were injected subcutaneously into nude mice. Mice were intraperitoneally injected with cisplatin (5 mg/kg) twice per week once the tumors were established. Tumor growth was monitored with a standard caliper. C In vivo bioluminescence imaging was shown on day 36. D PAK4 and GRP78 expression profiles were examined by western blot in xenograft tumors. E Representative IHC staining results of PAK4 and GRP78 were examined in xenograft tumors. F A positive correlation between PAK4 and GRP78 expression was observed by IHC staining in xenograft tumors. Data are expressed as mean ± standard deviation.

Fig. 6. PAK4 and GRP78 expression profiles in NSCLC tissues.

A Representative immunohistochemical staining (left panels) and statistical analysis (right panels) of GRP78 protein expression difference between 55 cisplatin-sensitive NSCLC tissues and 104 cisplatin-resistant NSCLC tissues. B The correlation between PAK4 expression and GRP78 expression in 159 human NSCLC tissues was examined by IHC staining. C The relative mRNA expression of the GRP78 in 20 cisplatin-sensitive NSCLC tissues and matched cisplatin-resistant NSCLC tissues was examined by Real-time PCR. D The correlation between relative PAK4 mRNA expression and relative GRP78 mRNA expression in 20 cisplatin-sensitive NSCLC tissues and matched cisplatin-resistant NSCLC tissues. Data are shown as the means of three independent experiments or representative data. Data are expressed as mean ± standard deviation. **P < 0.01 by Student’s t test.

Fig. 7. PAK4 knockdown inhibited GRP78 expression.

A PAK4, GRP78, p-MEK1, and MEK1 expression were examined by western blot. B Flow cytometry analyses of the percentages of apoptotic cells in A549-res and NCI-H520-res cells transfected with the shPAK4 or MEK1 plasmids or in combinations between shPAK4 and MEK1. C A549-res and NCI-H520-res cell lysates were immunoprecipitated with PAK4 antibody and subjected to Western blotting to ascertain MEK1 and PAK4 expression using indicated antibodies (upper panels). Cell lysates were immunoprecipitated with MEK1 antibody (lower panels). D A549-res cells and NCI-H520-res cells were treated with PD 98059 or control for 48 h, and PAK4, MEK1, p-MEK1, and GRP78 expression profiles were examined by western blot. E A549-res cells and NCI-H520-res cells were treated with MEK1 inhibitor (PD 98059). Luciferase activity of GRP78 was examined after 48 h. F Luciferase activity of GRP78 was examined in the overexpression of PAK4 cells (A549 and NCI-H520) with or without PD 98059. Data are shown as the means of three independent experiments or representative data. Data are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01 by Student’s t test.