Rottmann et al. 10.1073/pnas.0505114102. |
Fig. 8. HA1E or HA1E-MYC cells were treated with the indicated concentrations of LiCl or KCl for 8 h before induction of apoptosis as in Fig. 2. KCl served as a control for osmotic stress. Cells were treated with the indicated concentrations of DR5-A; 20 h later, viability was determined by CellTiterGlo assays. Error bars indicate standard deviations of triplicate measurements.
Fig. 9. Small-molecule inhibitors of GSK3 act as DR5-A synergists. (a) HA1E-MYC cells were treated with the indicated concentrations of the commercially available small-molecule GSK3 inhibitor GSK3-IX (6-bromoindirubin-3-oxime, catalog no. 361550, Calbiochem). Cells were simultaneously treated with the indicated concentrations of DR5-A; 20 h later, viability was determined by CellTiterGlo assays.(b) A Western blot of MYC levels in control versus LiCL or GSK3-IX treated cells is pictured to the right, and quantitation by densitometry is denoted below each band.
Fig. 10. GSK3b regulates sensitivity to DR5-mediated apoptosis via phosphorylation of MYC T58. (a and b) Mutation of the GSK3b MYC T58 phosphorylation site mimics GSK3b or FBW7 loss of function. Derivatives of HA1E cells expressing retrovirally transduced WT MYC (b) or the T58 phosphorylation site mutant MYCT58A (a) were transfected with the indicated small inhibitory RNAs (siRNAs) and treated with DR5-A and assayed for viability.
Fig. 11. Confirmation of small inhibitory RNA (siRNA) gene silencing and FBW7 deletion. (a) The efficacy of siRNAs targeting GSK3 isoforms and FBW7 in HCT116 cells was determined by Western blot analysis. siDR5 was validated by RT-PCR of DR5. The asterisk denotes a nonspecific immunoreactive product. (b) To confirm the endogenous status of FBW7 in the HCT116 deletion series, RT-PCR was performed using primers that lie in exon 5 and 9 such that the homozygous knockout of FBW7 results in no RT-PCR product (see Methods).
Fig. 12. HCT116 FBW7–/– cells were transfected with the indicated small inbitiory RNAs (siRNAs) and sensitivity to DR5-A was determined by viability assays as described for Fig. 5b.
Fig. 13. Schematic of FBW7 structure and position of the mutation in HT115 cells. HT115 cells are colorectal carcinoma-derived cancer cells that bear a heterozygous naturally occurring point mutation in the FBW7 gene. (a) The 11 exons of FBW7 and the two main structural motifs, the F-box and seven WD40 repeats, are shown. The point mutation lies in exon 8 within the third WD40 repeat, which is critical for substrate recognition by FBW7. The three reported isoforms of FBW7 (a, b, and g) differ in exon 1 but share all other exons. (b) HT115 colon carcinoma cells containing a naturally occurring heterozygous loss-of-function FBW7 mutation are highly sensitive to DR5 agonists, which can be partially reversed by stable transfection of a WT FBW7 cDNA (FBW7 clone 1 and FBW7 clone 2).
Fig. 14. FBW7 heterozygous mutant HT115 cells are highly sensitive to DR5-A and can be rescued from DR5-A-mediated apoptosis by MYC depletion. HT115 cells were transfected with small inhibitory RNAs (siRNAs) targeting MYC and cells were assayed for DR5-A-sensitivity. siDR5 was included as a control.
Fig. 15. Depletion or mutation of the FBW7 tumor suppressor enhances DR5-A sensitivity in tumor-derived cell lines in a MYC-dependent manner. Derivatives of HCT116 FBW7 WT and mutant cell lines expressing the dominant negative MYC protein MADMYC were generated by retroviral transduction and treated with DR5-A for 20 h followed by assessment of viable cells.
Fig. 16. FBW7 depletion increases formation of micronuclei, which can be rescued by siCyclinE but not by siMYC. (a–c) HA1E (a), HA1E-MYC (b), or HCT116 FBW7+/+ (c) cells were transfected with the siRNAs indicated, fixed 48 h later, and stained with DAPI. Pictures of 400–600 randomly chosen cells were taken, and their micronuclei were counted. Graphs show the number of micronuclei as a percentage of total cell number. (d) Same as in a–c but using untransfected HCT116 FBW7 WT and mutant cells.
Table 1. The top 14 hits of the screen listed by decreasing viability ratio (see Fig. 1b)
Accession no. | siRNA target name | Viability ratio HA1E/HA1E-MYC |
NM_002870 | RAB13, member RAS oncogene family | 3.40 |
NM_007218 | TRC8 patched related protein translocated in renal cancer | 2.71 |
AK091708.1 | Homo sapiens cDNA FLJ34389 fis, clone HCHON2000087 | 2.59 |
XM_048605.6 | H. sapiens similar to Ser/Thr-protein kinase NEK1 | 2.56 |
NM_022963 | H. sapiens FGFR4, transcript variant 2 | 2.48 |
NM_000264 | H. sapiens PTCH | 2.33 |
NM_003684 | H. sapiens MKNK1 | 2.22 |
NM_001258 | H. sapiens CDK3 | 2.12 |
NM_001982 | H. sapiens ERBB3 | 2.10 |
XM_114728.1 | H. sapiens similar to homeodomain-interacting protein kinase 1 (LOC204851) | 2.09 |
NM_004422 | DVL2 | 2.06 |
NM_001315 | H. sapiens MAPK14 | 2.03 |
NM_002757 | H. sapiens MAP2K5 | 2.02 |
NM_002093 | GSK3b: glycogen synthase kinase 3b | 2.01 |
Small inhibitory RNAs (siRNAs) that primarily sensitize MYC-expressing cells to death receptor 5-A yield higher viability ratios (HA1E/HA1E-MYC). NEK1, NimA-related protein kinase 1; FGFR4, fibroblast growth factor receptor 4; MKNK1, mitogen-activated protein kinase (MAPK)-interacting Ser/Thr kinase 1; PTCH, patched (Drosophila) homolog; CDK3, cyclin-dependent kinase 3; ERBB3, v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 3; DVL2, dishevelled 2 (homologous to Drosophila dsh); MAP2K5, MAPK kinase 5.
Supporting Methods
Cell Lines and Materials.
Genetically defined immortalized HA1E cells were obtained from Robert Weinberg (Whitehead Institute, Cambridge, MA) and grown in Minimum Essential Eagle’s Medium supplemented with glutamine, penicillin/streptomycin, and 10% FCS. The colon cancer cell line HCT116 and its FBW7+/– and FBW7–/– derivatives were obtained from Bert Vogelstein (The Johns Hopkins University, Baltimore) and grown in McCoy’s 5A medium supplemented as above. The colon cancer cell line HT115 was obtained from the European Collection of Cell Cultures and grown in DMEM supplemented as above. Antibodies for immunoblotting were obtained from the indicated suppliers: MYC-9E10 (Santa Cruz Biotechnology), T58-phosphoMYC (catalog no. 9401, Cell Signaling Technology), Caspase-8 (catalog no. 12F5, Biosource International, Camarillo, CA), FLIP (NF-6, Axxora, San Diego), FBW7 (Mobi-Tec, Gottingen, Germany), GSK3 (Stressgene Biotechnologies, Victoria, Canada), Cyclin E (HE12, Pharmingen), and DR5A [generated at The Genomics Institute of the Novartis Research Foundation (2)]. All siRNA smartpools (targeting GSK3a, GSK3b, Cyclin E, and MYC) were purchased from Dharmacon.Retroviruses.
Retroviruses were produced by Lipofectamine 2000-mediated (Invitrogen) transfection into Phoenix-A producer cells (Garry Nolan, Stanford University, Stanford, CA). Retroviral infections were performed by centrifuging target cells at 900 ´ g for 90 min at 25°C with 50% retroviral supernatant containing 20 mM Hepes and 8 mg/ml polybrene. Two rounds of infection were typically performed. Retroviral vectors used were LZRS-IRES-EGFP for MYC, pBABEpuro for MADMYC, and pWZLblast for MYC or MYC-T58A. Puromycin or blasticidin at 1 mg/ml was used for selection.Small inhibitory RNA (siRNA) Library Screening.
The siRNA collection was prepared and plated as described in ref. 1. The siRNA collection contains 624 siRNAs designed to specifically silence each of 380 known and predicted kinases, 100 genes of unknown function, and 144 known genes of interest including genes known to play a role in apoptosis pathways. siRNAs were arrayed in 384-well microtiter plates in duplicate at 8 ng per well. To transfect siRNA libraries into HA1E and HA1E-MYC cells, we used reverse transfections. Briefly, 10 ml of Opti-Mem/Lipofectamine 2000 (125:1) mixture was dispensed into each well to incubate with siRNAs followed by dispensing of 40 ml of cells. Cross-linked DR5-A (10 ml) was added 48 h later to reach a final concentration of 1 mg/ml. Another 24 h later, cell viability in each well was measured by CellTiter-Glo (Promega). For each 384-well plate, the signal was normalized by dividing each well by the average of 24 wells on the same plate containing siRNAs against Luciferase (siGL3) and multiplying by 100 to obtain normalized viability (percent viability). For each cell line, siRNAs that decrease viability by >20% (toxic siRNAs) were excluded from our analysis. For Fig. 1b, each spot represents the effects of an individual siRNA. The siRNAs were ranked in increasing order on the x axis by their ability to sensitize HA1E-MYC cells to DR5-A-induced apoptosis. The normalized viability of siRNA-transfected HA1E cells treated with DR5-A, divided by the normalized viability of similarly treated HA1E-MYC cells was plotted as a ratio on the y axis. Accordingly, siRNAs that strongly potentiate DR5-mediated apoptosis have high x-axis values, whereas those with high specificity for MYC-expressing cells have high y-axis values. The siRNA for GSK3b is highlighted.Antibodies.
For in vitro apoptosis assays, DR5-A was crosslinked by incubating with F(Ab′)2 fragment goat anti-mouse anti-Fcg (Jackson ImmunoResearch) for 1 h at room temperature at a 1:3 ratio by weight. Western blot analysis was performed as previously described (2).siRNA.
Individual siRNAs used were purchased from Qiagen (Valencia, CA): siGSK3a (GTG ATT GGC AAT GGC TCA T), siGSK3b-1 (GTA TTG CAG GAC AAG AGA T), siGSK3b-S (GC AAA TCA GAG AAA TGA AC), siFBW7-2 (GGG CAA CAA CGA CGC CGA A), siFBW7-3 (AAG GCA CTC TAT GTG CTT TCA), siMYC (CAC GTC TCC ACA CAT CAG CAC AA), and control siRNA siGL3 (directed against the luciferase gene from vector pGL3) AAC TTA CGC TGA GTA CTT CGA TT.Cells were transfected with 20 ng per well in 96-well plates or 600 ng per well in 6-well plates using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions and treated with DR5-A or prepared for FACS analysis, immunoblotting, or semiquantitative RT-PCR 48 h after transfection.
Semiquantitative RT-PCR.
Total RNA was extracted with an RNeasy Mini kit (Qiagen). RNA (1 mg) was reverse-transcribed by using the ThermalScript system (Invitrogen). cDNA (1 ml) was then used for PCR amplification with the following primers: FBW7_forward (AAG TTG GAC CAT GGT TCT GAG), FBW7_reverse (A CAC AGC GGA CTG CTG CAA C), DR5_forward (ATG GAA CAA CGG GGA CAG AAC), and DR5_reverse (TTA GGA CAT GGC AGA GTC TGC).FACS.
Cells were siRNA-transfected as above, and harvested with Accutase 48 h after transfection. The cells were then pelleted, incubated with 10 mg/ml DR5-A (mouse antibody) in Hank’s buffer (supplemented with 0.1% 0.5 M EDTA/0.1% 1M Hepes/1% PBS/0.1% sodium azide) for 90 min at 4°C, washed two times, incubated with 1:1,000 diluted allophycocyanin-crosslinked goat anti-mouse secondary antibody (Molecular Probes) for 30 min, washed again, and analyzed by FACS.Viability Assays.
Cells (8,000 to 10,000 per well) were plated in 96-well plates in the appropriate cell culture medium, incubated with LiCl or KCl for 8 h or transfected with siRNAs, and treated with DR5-A 48 h after transfection for an additional 20 h. Cell viability was measured in triplicate by CellTiterGlo (Promega) according to the manufacturer’s instructions.FBW7
Rescue Experiments. HT115 cells were transfected with pCMV6-FBW7 coding for full-length FBW7g by using Fugene 6 transfection reagent (Roche). pBABEpuro was cotransfected at a 1:4 ratio by weight allowing for selection with 2 mg/ml puromycin. Stable cells were plated in 96-well plates to attach overnight and treated with DR5-A for 20 h. The cell viability assay was performed subsequently.Animal Experiments.
HCT116 FBW7+/+ or FBW7+/– cells (3 × 106) were implanted by s.c. injection into female, 8-week-old BALB/c nude mice using a 27-gauge needle. For each group, tumors formed in 8 of 8 animals and were measured with calipers in three dimensions. Tumor volumes were calculated with the formula (L ´ W ´ H)/2. When tumors reached a mean volume of 80 mm3, mice were rank-ordered by tumor volume with the smallest and largest tumor excluded and then divided into even-rank and odd-rank groups for i.p. injection with 100 mg of DR5-A or vehicle control (50 mM sodium citrate, pH 7.0/140 mM NaCl). Mice were treated every other day for a total of seven times. All animal husbandry and experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Genomics Institute of the Novartis Research Foundation.1. Aza-Blanc, P., Cooper, C. L., Wagner, K., Batalov, S., Deveraux, Q. L. & Cooke, M. P. (2003) Mol. Cell 12, 627–637.
2. Ren, Y. G., Wagner, K. W., Knee, D. A., Aza-Blanc, P., Nasoff, M. & Deveraux, Q. L. (2004) Mol. Biol. Cell. 15, 5064–5074.