P21-activated kinase 1 regulates resistance to BRAF inhibition in human cancer cells

Abstract

BRAF is a commonly mutated oncogene in various human malignancies and a target of a new class of anti-cancer agents, BRAF-inhibitors (BRAFi). The initial enthusiasm for these agents, based on the early successes in the management of metastatic melanoma, is now challenged by the mounting evidence of intrinsic BRAFi-insensitivity in many BRAF-mutated tumors, by the scarcity of complete responses, and by the inevitable emergence of drug resistance in initially responsive cases. These setbacks put an emphasis on discovering the means to increase the efficacy of BRAFi and to prevent or overcome BRAFi-resistance. We explored the role of p21-activated kinases (PAKs), in particular PAK1, in BRAFi response. BRAFi lowered the levels of active PAK1 in treated cells. An activated form of PAK1 conferred BRAFi-resistance on otherwise sensitive cells, while genetic or pharmacologic suppression of PAK1 had a sensitizing effect. While activation of AKT1 and RAC1 proto-oncogenes increased BRAFi-tolerance, the protective effect was negated in the presence of PAK inhibitors. Furthermore, combining otherwise ineffective doses of PAK- and BRAF-inhibitors synergistically affected intrinsically BRAFi-resistant cells. Considering the high incidence of PAK1 activation in cancers, our findings suggests PAK inhibition as a strategy to augment BRAFi therapy and overcome some of the well-known resistance mechanisms.

Keywords: AZD6244; MAP Kinase Cascade; PF3758309; melanoma; vemurafenib.

Figure 1. The effects of inhibition of PAK on RAC1-mediated resistance to BRAFi

(A) A375 cells were transduced with RAC1 P29S and shPAK1 (“RAC1+shPAK1”), pLX304 empty vector and shPAK1 (“shPAK1”), RAC1 P29S and a non-targeting “scrambled” shRNA (“RAC1”), or pLX304 and a “scrambled” shRNA (“Control”). The cells were treated for five days with the indicated doses of PLX4720 in quadruplicates, and the numbers of remaining cells were compared by methylene blue staining extraction method. The values with standard deviations are plotted as percentages of those in the corresponding cultures treated with the vehicle (DMSO) alone. (B) The IC₅₀ concentrations were calculated for the data shown in (A) and are presented relatively to that of the “Control” culture. The error bars denote 95% confidence intervals. (C) The indicated cell lines were treated with 100nM PLX4720 or DMSO, and the corresponding lysates were probed by immunoblotting for the expression total and activated (phosphorylated) PAK1 and ERK1/2, as well as α-tubulin (loading control). (D) A375 cells transduced with RAC1-P29S (“RAC1”) or pLX304 (“Control”) were treated for 5 days with 50nM vemurafenib (“VEM”), 2.5uM IPA3 (“IPA”) or 10nM PF3758309 (“PF”), alone or in the indicated combinations. The numbers of remaining cells were compared using the methylene blue straining and extraction method and are shown as a fraction of the values in the corresponding “Untreated” (exposed to DMSO alone) cultures. (E) The lysates from the cells treated for 48h as in (D) were probed by immunoblotting for the levels of α-tubulin (loading control) and the activated (phosphorylated) forms of PAK1 and ERK1/2.

Figure 2. Constitutive activation of PAK1 reduces the effects of BRAF inhibition

(A) A375 cells transduced with PAK1 T423E (“caPAK1”) or the corresponding empty vector (“Control”) were treated in quadruplicates with the indicated doses of PLX4720. The numbers of remaining cells were compared using the methylene blue staining and extraction method. The values with standard deviations are plotted as percentages of those in the corresponding cultures treated with the vehicle (DMSO) alone. (B) The IC₅₀ concentrations were calculated for the data shown in (A) and are presented relatively to that of the “Control” culture. The error bars denote 95% confidence intervals. (C) A375 cells transduced with PAK1 T423E (“caPAK1”) or the corresponding empty vector (“EV’) cells were treated for 48h with 60nM of PLX4720 or the corresponding vehicle (DMSO) alone. Cell lysates were probed by immunoblotting for GAPDH (loading control) and total and activated (phosphorylated) MEK1/2 and ERK1/2. (D) A375 transduced with caPAK1 or the corresponding empty vector (“Control”) were treated for 48h with PLX4720 or DMSO. EdU (10uM) was added and 1h later the cells were then stained as described in Materials and Methods. Representative images are shown. (E) For the cells treated as in (D), the fraction of EdU-labeled cells was scored on 5 randomly chosen fields of view. The bars show the values for PLX470-treated cultures as a fraction of the corresponding DMSO-treated controls. The error bars represent standard deviations.

Figure 3. The effects of inhibition of PAK on AKT-mediated resistance to BRAFi

(A) A375 cells were transduced with activated AKT and PAK1 shRNA (“AKT+shPAK1”), pLM-CMV-neo empty vector and PAK1 shRNA (“shPAK1”), activated AKT and a non-targeting “scrambled” shRNA (“AKT”), or pLM-CMV-neo and a “scrambled” shRNA (“Control”). The cells were treated in quadruplicates with the indicated doses of PLX4720. The numbers of remaining cells were compared using the methylene blue staining and extraction method. The values with standard deviations are plotted as percentages of those in the corresponding cultures treated with the vehicle (DMSO) alone. (B) The IC50 concentrations were calculated for the data shown in (A) and are presented relatively to that of the “Control” culture. The error bars denote 95% confidence intervals. (C) A375 cells transduced with activated AKT (“AKT”) or the corresponding vector control (“control”) were treated with 40_nM PLX4720 or/and 8_nM PF-3758309 over 5 days. The numbers of remaining cells were compared using the methylene blue staining and extraction method and are plotted after normalization to the respective drug-free populations (“Untreated”). The error bars denote standard deviations.

Figure 4. Synergism between BRAF and PAK inhibitors in BRAFi-resistant cells

(A) B-CPAP cells were treated for 5 days with the indicated doses of PLX4720. The numbers of remaining cells were compared by methylene blue staining and extraction method and are plotted relatively to those in untreated populations. (B) B-CPAP cells were treated for 5 days with PLX4720 (500nM; “PLX4720”) or PF3758309 (6nM; “PF”) or a combination thereof (“Combo”). The numbers of the remaining cells were compared by the methylene blue staining and extraction method and are shown relatively to those in untreated populations. (C) Combinational Index values were calculated using the Chou-Talalay method, as described in Materials and Methods, from a series of experiments conducted on B-CPAP cells essentially as in (B) and using the indicated doses of the drugs. Combinational Index values below 1 indicate synergy between the compounds. (D) HT29 cells were treated for 5 days with the indicated doses of vemurafenib. The numbers of remaining cells were compared by methylene blue staining and extraction method and are plotted relatively to those in untreated populations. (E) HT29 cells were treated for 5 days with vemurafenib (250nM; “VEM”) or PF3758309 (12nM; “PF”) or a combination thereof (“VEM/PF”). The numbers of the remaining cells were compared by the methylene blue staining and extraction method and are shown relatively to those in untreated populations. (F) Combinational Index values were calculated using the Chou-Talalay method, as described in Materials and Methods, from a series of experiments conducted on HT29 essentially as in (E) and using the indicated doses of the drugs. The values are shown as averages ± standard deviation from three independent experiments.