Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents.

PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n = 980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro . Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting.

Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0564-5) contains supplementary material, which is available to authorized users.

Materials and methods

Materials, cell culture and viability assays FRAX1036 was synthesized by Afraxis, Inc. (La Jolla, CA, USA) and docetaxel was purchased from Selleck Chemicals (Houston, TX, USA). Antibodies used for immunoblotting (p-MEK1-S298, p-CRAF-S338, Cleaved PARP, Cyclin D1, p-Stathmin-S16, p-β-catenin-S675, MCL-1, BCL-xL, p-Bad-S112 and PAK1) were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-Actin was purchased from Sigma (St Louis, MO, USA). Cell lines were acquired from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained at 37°C and 5% CO 2 in RPMI 1640 media with 10% fetal bovine serum and 2 mM L-glutamine. U2OS-red fluorescent protein (RFP)-Tubulin cells (Marinpharm, Luckenwalde, Germany) were stably transduced with a plasmid expressing green fluorescent protein (GFP)-histone H2B. Cell transfections and treatments were performed using short interfering RNA oligonucleotides for PAK1 from Dharmacon RNAi Technologies (Chicago, IL, USA). Cellular viability was assessed via ATP content using the CellTiter-Glo Luminescent Assay (Promega, Madison, WI, USA) and results represent mean ± standard deviation from three experiments. PAK1/CCND1 survival analysis Breast tumors from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset [ 15 ] with survival and DNA copy number data were selected, yielding 980 patients. DNA copy number was calculated using Affymetrix SNP6.0 arrays and a modified version of the PICNIC algorithm [ 19 ], published recently [ 20 ]. Samples were identified as having amplification of either PAK1 or CCND1 if the absolute copy number of the respective gene was >5 copies. The Kaplan-Meier plot and log-rank test were performed using the censored survival values (days since diagnosis) provided with the METABRIC dataset and our calculated PAK1 amplification status using the R language [ 21 ], version 3.1, and the R package “survival”, version 2.37-7. A Cox proportional hazard model was constructed using the METABRIC censored survival data, Nottingham prognostic index (NPI), patient age, and patient PAM50 breast cancer subtype classification in addition to the interaction of CCND1 and PAK1 amplification statuses. More specifically, the model “survival ~ NPI + age + PAM50 + CCND1 * PAK1 ” was fit using the “coxph” R package, where ccnd1 and pak1 are binary variables, as discussed above. The forest plot was produced using the coefficients from this model and their P -values. The whiskers on this plot represent ±1.96 × the standard error for each coefficient. The coefficient for amplification of both CCND1 and PAK1 (dual amplification) in the same sample was calculated as the sum of the coefficients “pak1Amplified”, “ccnd1Amplified”, and the coefficient for the interaction term for these two terms.

Bliss analysis

Cellular viability was assessed via ATP content using the CellTiter-Glo Luminescent Assay (Promega, Fitchburg, WI, USA) after a 4-day incubation period, and results represent mean ± standard deviation from three experiments. Total luminescence was measured on a Wallac Multilabel Reader (Perkin-Elmer, Waltham, MA, USA). Cells were treated simultaneously with FRAX1036 (dose range = 0 to 5 μM) or docetaxel (dose range = 0 to 0.4 nM) in an 8 × 10 matrix of concentrations. Combination synergy of FRAX1036 and docetaxel was determined by Bliss independence analyses. A Bliss expectation for a combined response C was calculated by the equation: C = (A + B) - (A × B) where A and B are the fractional growth inhibitions of given doses of drug A and B. ΔBliss scores were summed across the dose matrix to generate a Bliss sum. Bliss sum = 0 indicates that the combination effect is additive while Bliss sum >0 indicates synergy effect and Bliss sum