U251 human glioblastoma cells were purchased from Sigma-Aldrich (St. Louis, MO, USA) and the U87 and A172 human glioblastoma cells were from the American Type Culture Collection (ATCC; Manassas, VA, USA). Normal human astrocytes were obtained from Sciencell Research Laboratories (Carlsbad, CA, USA). All cell lines were maintained in Dulbecco's modified Eagle's medium-DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen), 2 mM L-glutamine (Invitrogen), 100 nM MEM non-essential amino acids (Invitrogen) and penicillin-streptomycin (Invitrogen) at 37°C and 5% CO₂. Accell SMARTpool siRNAs targeting MACF1 were purchased from GE Dharmacon (Chicago, IL, USA). Temozolomide was purchased from Sigma-Aldrich. GBM76 patient derived xenograft cells were established at the Mayo Clinic (Rochester, MN, USA) (14), while primary xenograft lines T4105 and T4302 were provided by Jeremy Rich from the Cleveland Clinic (Cleveland, OH, USA). Patient derived xenograft cell lines (GBM76, T4105 and T4302) were serially passaged as subcutaneous tumors in immunocompromised mice and maintained as ex vivo cultures in neurobasal medium supplemented with B27 serum substitute, 20 ng/ml EGF and 20 ng/ml bFGF (Life Technologies, Carlsbad, CA, USA).

Brain tumor tissue arrays were purchased from US Biomax, Inc., (Rockville, MD, USA) to evaluate MACF1 expression. Each slide contained the following number and array of brain tumor tissue sections provided by the supplier: 10 grade (I–II) astrocytomas, 10 grade (III–IV) astrocytomas, 33 grade (IV) glioblastomas, 3 oligoastrocytomas, 6 grade (I–II) oligodendrogliomas, 3 medulloblastomas, 3 anaplastic oligodendrogliomas, 4 ependymomas and cancer adjacent normal tissue from 5 brains. Paraffin-embedded tissue sections were dewaxed in xylene for 5 min, rehydrated in 100% ethanol, 95% ethanol and water. Next tissue was fixed with 100% methanol at −20°C for 15 min and washed three times with phosphate-buffered saline (PBS). Detection of MACF1 was conducted using the ImmunoCruz rabbit ABC Staining system (Santa Cruz Biotechnology, Dallas, TX, USA). Tissue sections were blocked for 1 h in 1.5% normal horse serum in PBS at room temperature, permeabilized with 0.05% Triton-X in PBS for 10 min, incubated overnight at 4°C with a polyclonal MACF1 antibody (Santa Cruz Biotechnology) and rinsed three times with PBS. Subsequently, sections were incubated with a biotinylated secondary antibody for 3 h at room temperature and washed in PBS three times 5 min each. Sections were then incubated with an avidin-biotinylated HRP complex for 30 min, rinsed again three times with PBS and MACF1 protein visualized as diaminobenzidine (DAB) positive cells in tissue sections with a peroxidase substrate solution containing substrate buffer, DAB and peroxidase substrate according to the manufacturer's instructions. Images were then taken with a Leica DM4000 microscope and QImaging system (Leica Microsystems, Inc., Buffalo Grove, IL, USA).

For silencing experiments 3.5×10⁴ U251, A172, or patient derived xenograft cells were plated in 6-well plates, grown for two days to 70% confluency, and treated with 1 µM non-targeting control siRNAs, 1 µM siRNAs targeting MACF1 (GE Dharmacon), or shRNAs targeting MACF1 from the MISSION shRNA Library (TRCN0000294117 and TRCN0000294123; Sigma-Aldrich) and allowed to incubate for 6 days in Accell media for siRNAs or neurobasal medium for shRNAs. MACF1 expression was subsequently examined in control and MACF1 silenced cells using immunoblotting procedures.

To assess the effects of silencing MACF1 on cell proliferation and viability, cells were treated with 1 µM of non-targeting control siRNAs or siRNAs targeting MACF1 (GE Dharmacon) and allowed to incubate for 6 days in 6-well plates as described above. At the end of the 6-day incubation period 1.5×10⁴ cells treated with control or siRNAs targeting MACF1 were replated in 12-well plates and allowed to incubate in the absence of siRNAs for 2, 4, and 6 days in DMEM media (Invitrogen) containing 10% FBS (Invitrogen). At the end of each time-point cells were evaluated using the crystal violet cell proliferation assay, one of three standard assays that can be used to evaluate cytotoxicity and/or cell viability that provide a measure of cell proliferation (15). Tissue culture medium was removed and the cell monolayer was fixed with 100% methanol for 5 min and stained with 0.5% crystal violet in 25% methanol for 10 min. Cells were then washed three times 5 min each with distilled water to remove excess dye and allowed to dry overnight at room temperature. The incorporated dye was then solubilized in 0.1 M sodium citrate (Sigma-Aldrich) in 50% ethanol. Next, 100 µl of experimental samples were transferred to 96-well plates and optical densities read at 540 nm using a xMark microplate absorbance spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA). For combinatorial experiments glioblastoma cells were also first treated for 6 days with control non-targeting siRNAs or siRNAs targeting MACF1, as previously described. Next 2.5×10⁴ siRNA treated cells were replated in 12-well plates in the absence of siRNAs. Controls and conditions for combinatorial experiments included: replated siRNA treated cells grown for 72 h, replated siRNA treated cells exposed to TMZ or DMSO 6 h after replating and allowed to incubate in TMZ or DMSO for 72 h, and non-treated siRNA cells exposed to TMZ or DMSO 6 h after plating and grown for 72 h in the presence of TMZ or DMSO. DMSO is a solvent used to dissolve drugs and was used as a vehicle control to discern the effects of TMZ from the drug solvent. To evaluate the combinatorial effects of silencing MACF1 and TMZ treatment on cell proliferation and viability the cell proliferation assay, as described above, was also used at the end of the 72-h incubation time-point.

Motility assays were conducted according to the manufacturer's instructions (Cell Biolabs, Inc., San Diego, CA, USA). MACF1 silencing was performed as described above. Cell suspensions containing 0.5–1.0×10⁶ cells/ml of siRNA control or siRNA targeted MACF1 treated cells were prepared in serum-free media, while 500 µl of media containing 10% FBS was added to the lower chamber of the migration plate. A total of 300 µl of siRNA treated cell suspensions were then added to the inside of each insert and were allowed to incubate for 24 h at 37°C and 5% CO₂. Subsequently, non-migratory cells were removed from plate inserts (per manufacturer's instructions), migratory cells were stained with crystal violet and the dye eluted as described above. Optical densities were read at 595 nm using a xMark microplate absorbance spectrophotometer (Bio-Rad Laboratories) and statistical evaluation of the data was performed as described below.

Cells treated for 6 days with control siRNAs or siRNAs targeting MACF1 were rinsed with PBS and lysed with CelLytic M Cell lysis reagent (Sigma-Aldrich). Protein concentrations were subsequently determined using the Bradford method. Proteins were separated by SDS-PAGE in 4-15% polyacrylamide gels (Bio-Rad Laboratories), then transferred to nitrocellulose membranes. For immunoblotting, nitrocellulose membranes were incubated with MACF1 (Santa Cruz Biotechnology), Axin1 (Cell Signaling Technology, Danvers, MA, USA), phospho β-catenin (Cell Signaling Technology) and GAPDH (Cell Signaling Technology) antibodies recognizing target proteins overnight at 4°C. The membranes were then incubated with an HRP-conjugated secondary antibody for 1 h at room temperature, analyzed by enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific, Nashville, TN, USA) and visualized using a UVP BioSpectrum imaging system (UVP, LLC, Upland, CA, USA). Densitometry analysis was performed with the Image Studio Lite software (LI-COR Biosciences, Lincoln, NE, USA).

Cell proliferation, migration, time-course and combinatorial experiments were each performed at least three times with duplicate or triplicate samples. Means were determined by averaging duplicate or triplicate samples within each independent experiment. Students t-tests and ANOVA analysis were used to evaluate the significance between the experimental conditions with a P-value of 0.05 and 0.01, respectively.

Identification and expression profiling of tumor specific biomarkers are essential for clinical diagnostic and prognostic purposes. Using immunohistochemistry procedures we examined MACF1 expression in low and high grade brain tumors. Our data showed that MACF1 was expressed diffusely and at the periphery in 40% of grade IV glioblastoma tissue sections as compared to its absence in normal brain tissue (Fig. 1A–C). In contrast to its expression in glioblastoma, MACF1 was absent in lower grade brain tumors (oligodendroglioma and medulloblastoma) and normal brain tissue (Fig. 1D–F), but present in grade II–IV astrocytomas as compared to normal brain tissue (Fig. 2). Complementing data observed in human tissue, immunoblotting procedures of MACF1 in glioblastoma cell lines also displayed significantly higher levels of MACF1 as compared to its expression in normal human astrocyte cells (Fig. 2). Collectively, these data provide evidence that MACF1 is predominately expressed in high grade malignant brain tumors and suggest that it is a potential biomarker and target in these cancers.

MACF1 to date has received no consideration as a target in cancer and associated cell behavior. Using siRNAs to inhibit MACF1 function we assessed the effects of downregulating MACF1 expression on glioblastoma cell proliferation and migration. Western blot experiments demonstrated the efficacy of the siRNAs in silencing MACF1 in U251 and A172 glioblastoma cells. Our data also revealed that silencing MACF1 in U251 glioblastoma cells caused a 45 and 37% decrease in cell viability 4 and 6 days post-treatment, respectively (Fig. 3). However, suppressing MACF1 expression did not reduce A172 glioblastoma cell viability (Fig. 3). We further evaluated the effects of inhibiting MACF1 in patient derived xenograft cell lines (Fig. 4). Time course analysis displayed that shRNAs targeting MACF1 significantly inhibited the growth of T4105 and T4302 xenograft cell lines, while SF295 and GBM76 cells were decreased to a much lesser extent (Fig. 4). Consistent with the anti-proliferative effects of downregulating MACF1 protein levels in U251 cells, repression of MACF1 also significantly reduced U251 cell migration but not A172 cells (Fig. 5). Furthermore, because MACF1 has been implicated as a modulatory signaling contributor of the Wnt signaling pathway (16,17), whose dysregulation is well known to be involved in tumor proliferation and migratory invasion in a number of human cancers (18–20) including malignant brain tumors (21–24) we evaluated the impact of silencing MACF1 on Wnt-signaling proteins. Our data showed that downregulation of MACF1 protein levels was accompanied by decreased Axin1 and activated form of β-catenin protein levels (Fig. 6), suggesting that reduction of these Wnt-signaling mediators contribute to the anti-tumorigenic response of impairing MACF1 in glioblastoma.

Chemotherapeutic agents used to treat glioblastoma have been minimally effective in part as a consequence of expressed genetic aberrations that contribute to intrinsic and acquired tumor resistance (25). We subsequently performed combinatorial studies to test the effectiveness of downregulating MACF1 along with the effects of the alkylating agent, temozolomide (TMZ) in glioblastoma cells. For combinatorial experiments, glioblastoma cells were treated with siRNAs targeting MACF1 and 100 µM TMZ and compared to cells treated with TMZ alone or cells treated with siRNAs targeting MACF1 alone. ANOVA analysis (P<0.01) of data from cell proliferation assays revealed that the combination of MACF1 and TMZ treatment had the strongest effect on decreasing glioblastoma cell viability when compared to other treatment conditions and controls. Additionally, concurrent treatment of U251 glioblastoma cells with MACF1 targeting siRNAs and TMZ exhibited a 1.5- and 2-fold cell viability decrease as compared to cells treated with either MACF1 siRNAs or TMZ alone (Fig. 7). A similar response was observed in A172 cells, which displayed a 1.32- and 1.48-fold decrease in cell viability when treated concurrently with TMZ and siRNAs targeting MACF1 as compared to treatment with either of these conditions alone. We further examined by western blotting the effects of TMZ on the expression levels of MACF1 and Axin1 in U251 cells treated for 3 h with 100 µM TMZ. These experiments showed that TMZ increased the protein levels of MACF1 and the Wnt-cytoplasmic complex protein, Axin1 in U251 cells (Fig. 8), further supporting the combinatorial approach and need to inhibit MACF1 in glioblastoma cells treated with TMZ to achieve a synergistic effect.