Small Molecule Inhibition of Group I p21-Activated Kinases in Breast Cancer Induces Apoptosis and Potentiates the Activity of Microtubule Stabilizing Agents

Christy C Ong; Sarah Gierke; Cameron Pitt; Meredith Sagolla; Christine K Cheng; Wei Zhou; Adrian M Jubb; Laura Strickland; Maike Schmidt; Sergio G Duron; David A Campbell; Wei Zheng; Seameen Dehdashti; Min Shen; Nora Yang; Mark L Behnke; Wenwei Huang; John C McKew; Jonathan Chernoff; William F Forrest; Peter M Haverty; Suet-Feung Chin; Emad A Rakha; Andrew R Green; Ian O Ellis; Carlos Caldas; Thomas O'Brien; Lori S Friedman; Hartmut Koeppen; Joachim Rudolph; Klaus P Hoeflich

Disclosures

Breast Cancer Res. 2015;17(59) 

In This Article

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% CO2 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 <0 indicates antagonism effect. Statistical analysis comparing the Bliss sums for each cell line was conducted by the Student's t test.

Biochemical Assays

The activity/inhibition of human recombinant PAK1 (kinase domain), PAK2 (full length) or PAK4 (kinase domain) was estimated by measuring the phosphorylation of a FRET peptide substrate (Ser/Thr19) labeled with Coumarin and Fluorescein using Z'-LYTE™ assay (Invitrogen, Carlsbad, CA, USA). The 10 μL assay mixtures contained 50 mM HEPES (pH 7.5), 0.01% Brij-35, 10 mM MgCl2, 1 mM EGTA, 2 μM FRET peptide substrate, and PAK enzyme (20 pM PAK1; 50 pM PAK2; 90 pM PAK4). Incubations were carried out at 22°C in black polypropylene 384-well plates (Corning Costar, Corning, NY, USA). Prior to the assay, enzyme, FRET peptide substrate and serially diluted test compounds were preincubated together in assay buffer (7.5 μL) for 10 minutes, and the assay was initiated by the addition of 2.5 μL assay buffer containing 4× ATP (160 μM PAK1; 480 μM PAK2; 16 μM PAK4). Following the 60-minute incubation, the assay mixtures were quenched by the addition of 5 μL of Z'-LYTE™ development reagent, and 1 hour later the emissions of Coumarin (445 nm) and Fluorescein (520 nm) were determined after excitation at 400 nm using an Envision plate reader (Perkin Elmer). An emission ratio (445 nm/520 nm) was determined to quantify the degree of substrate phosphorylation.

Immunoblotting

Protein extracts were prepared at 4°C with RIPA Lysis Buffer (EMD Millipore Corporation, Billerica, MA, USA), 1 mM phenylmethylsulphonyl fluoride, Phosphatase Inhibitor Cocktail 2/3 and protease inhibitor cocktail (Sigma-Aldrich). For Western blot analysis, proteins were resolved by 4 to 12% SDS-PAGE and transferred to nitrocellulose membranes (Life Technologies, Grand Island, NY, USA). Immunoblotting was performed using the indicated primary antibodies and analyzed using secondary antibodies for enhanced chemiluminescence.

IncuCyte Apoptosis Assays

For caspase 3/7 activation apoptosis assays, cells were plated at 10,000 cells/well in 96-well Corning plates for 24 hours prior to treating with DMSO, FRAX1036, and/or docetaxel. Caspase 3/7 reagent was added at a 1:1000 dilution (Essen Bioscience No. 4440, Ann Arbor, MI, USA). Cells were imaged at 10× magnification in an IncuCyte Zoom Live-content imaging system (Essen Bioscience) at 37°C, 5% CO2. Images were acquired every 2 hours or 4 hours for 36 to 72 hours, two images/well. Data was analyzed using IncuCyte analysis software to detect and quantify green (apoptotic) cells/image. Each condition was performed in triplicate. Averages with SEM at each time point were plotted in Excel (Microsoft, Redmond, WA, USA). A t-test was performed for the final time point comparing the combination of FRAX1036 and docetaxel with each single agent in Prism (Graphpad, La Jolla, CA, USA). The apoptotic index was calculated from the apoptosis assays by dividing the final apoptotic cell count by the total cell count. Averages with SEM were plotted in Excel (Microsoft), and a t-test was performed comparing the combination of FRAX1036 and docetaxel with each single agent in Prism (Graphpad).

Live-cell Microscopy and Image Analysis

U2OS cells stably expressing GFP-Histone H2B and RFP-Tubulin were cultured in Dulbecco's modified Eagle's high glucose medium, 10% fetal bovine serum, NEAA, at 37°C and 5% CO2. For live imaging experiments, U2OS cells were plated in a 24-well glass bottom, black-walled plate (Sensoplate #662892, Greiner Bio-one, Monroe, NC, USA). On the following day, cells were treated with DMSO, FRAX1036, and/or docetaxel and imaged every 10 minutes for 72 hours with a 40× ELWD Plan Fluor objective (NA: 0.6, Nikon, Tokyo, JP) at 37°C and 5% CO2. Imaging was performed on a Nikon Ti-E perfect focus inverted microscope equipped with a spinning disk confocal CSU-X1 (Andor, Oxford Instruments, Abingdon, Oxfordshire, UK), motorized X,Y stage (Nikon), environmental chamber (OkoLab, Burlingame, CA, USA) and iXon3 897 EMCCD camera or Clara interline CCD camera (Andor, Oxford Instruments), all controlled by NIS-Elements software (Nikon, Tokyo, JP). Time-lapses were analyzed in NIS-Elements, and supplemental movies were generated in Quicktime Pro (Apple, Cupertino, CA, USA). For high-resolution imaging of microtubule organization, U2OS cells were imaged after 20 hours treatment with a 60× Plan Apo objective (NA: 1.4, Nikon). For immunofluorescence, MDA-MB-175 cells were methanol fixed, permeabilized in TBS-0.5% TritonX-100 and blocked in 2% bovine serum albumin, 0.1% Triton X-100. Microtubules were probed with primary antibody rat-anti tubulin (1:250, Serotec clone YL1/2), secondary antibody Alexafluor488 anti-rat (1:500, A-11006, Life Technologies, Grand Island, NY, USA), and mounted with Prolong Gold with DAPI (Life Technologies, Grand Island, NY, USA).

For the duration of mitosis/mitotic arrest and cell fate measurements, cells were monitored from the time they began to round up from the plate to the time when they were observed to divide, slip out of mitosis with micronuclei, or apoptose. Observations were made using the phase morphology of the cells as well as chromosome condensation/decondensation and mitotic spindle morphology in the fluorescent channels. Cells that divided or slipped were monitored for the remainder of the 72-hour movie, and subsequent cell events were recorded. Duration of mitosis/mitotic arrest was graphed in Prism (GraphPad), and significance was determined by one-way analysis of variance with multiple comparisons to compare each condition to one another. A t-test was performed on the two significant but close conditions (docetaxel and FRAX1036 + docetaxel).

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