Article Figures & Data
Figures
- Figure 1.
PDK1 and PI3K contribute to GPCR-mediated EGFR phosphorylation and HNSCC growth. PCI-37A (A), UM-22A (B), and PCI-6B (C) cells were transfected with nontargeting control (NTC) or PDK1 siRNA followed by 72 hours of serum starvation and stimulation with EGF (10 ng/mL), PGE2 (10 nmol/L), BK (10 nmol/L), or LPA (10 μmol/L) for 5 minutes. Cell lysates were collected and immunoprecipitated with EGFR and immunoblotted with an anti-phosphotyrosine antibody (P-EGFR) or total EGFR antibody, respectively. PDK1 levels were determined by Western blotting with an anti-PDK1 antibody. Densitometric analysis was done to determine cumulative results. The experiments were repeated 3 to 4 times with similar findings (P = 0.048, P = 0.0001). D, UM-22A cells were transiently transfected with PDK1 or NTC siRNA, serum starved for 48 hours, followed by stimulation with PGE2 (10 nmol/L) or BK (10 nmol/L) or vehicle for 24 hours. Percentage increase in proliferation was determined by trypan blue dye exclusion assay. Results were graphed using GraphPad Prism Software. The experiment was conducted twice in triplicate with similar results (P = 0.046, P = 0.007).
- Figure 2.
Kinase-dead PDK1 abrogates GPCR-mediated signaling and HNSCC growth. A, 1483 cells were stably transfected with pcDNA3.1 (VC) or pcDNA3-PDK1M (K110Q-kinase dead) and selected with neomycin for 2 weeks. Lysates were resolved by SDS-PAGE and probed for myc-tag and β-tubulin. B, 1483 vector-transfected control (VC) and 1483 kinase-dead PDK1 (PDKM) cells were seeded, serum-starved for 72 hours and stimulated with BK (10 nmol/L) for 5 minutes. Lysates were collected and immunoprecipitated with anti-phosphotyrosine antibody (P-EGFR) and immunoblotted for EGFR. Lysates were separately immunoblotted for EGFR to determine equal EGFR expression levels. Experiment was conducted twice with similar results 1483 VC and PDKM cells (C) were seeded and assessed for growth by trypan blue dye exclusion on days 1, 3, and 5. Cell counts were graphed using GraphPad Prism Software (Day 5, P = 0.039). IB, immunoblotting; IP, immunoprecipitation.
- Figure 3.
PDK1 contributes to EGFR-independent p70S6K phosphorylation. A, HNSCC (1483) cells were transfected with NTC, EGFR, PDK1, or a combination of EGFR and PDK1 siRNA for 72 hours. Lysates collected were analyzed for phospho-p70S6K, p70S6K, PDK1, and β-tubulin. The results are representative of 3 independent experiments. B, 1483 cells were stably transfected with pcDNA3 (VC) or pcDNA3-PDKM (kinase-dead). 1483 VC and PDKM cells were transfected with NTC or EGFR siRNA for 72 hours. Lysates were collected and resolved by SDS-PAGE. The results are representative of 2 experiments.
- Figure 4.
AR-12 inhibits in vitro and in vivo HNSCC growth, AKT phosphorylation, and cyclin D1. A, chemical structure of AR-12. B, HNSCC (PCI-37A, UM-22B, PCI-6B, and 1483) and normal mucosal epithelial cells (Het-1A) were treated with increasing concentrations of AR-12. After 72 hours, MTT assay was carried out and the IC50 values were determined using Prism (GraphPad) Software. The experiment was repeated 3 times with similar results. C, UMSCC-1 cells (3 × 106) were inoculated into the flanks of athymic nude mice. After the formation of tumor nodules (7 days later), mice were divided into 2 groups (9 mice per group), vehicle and AR-12 (100 mg/kg daily by oral gavage). Tumors were measured 3 times weekly over 28 days and graphed using GraphPad Prism (P < 0.0001, day 28). D, PCI-37A and UM-22B cells were treated with increasing concentrations of AR-12 for 72 hours. Lysates were assessed by immunoblotting for phospho-Akt, total Akt, and β-tubulin. Representative results from 3 independent experiments are shown.
- Figure 5.
AR-12 induces proapoptotic signaling. A, PCI-37A (left), UM-22B (right), and HET-1A (B) cells were treated with increasing concentrations of AR-12 for 36 hours and analyzed by immunoblotting for cleaved PARP (116 and 89Kd bands). Representative results from 3 independent experiments are shown. C, PCI-37A (left) and UM-22B (right) cells were treated with AR-12 and assessed for survivin expression by immunoblotting. The experiment was conducted twice with similar results.
- Figure 6.
PDK1 targeting enhances EGFR inhibition in HNSCC. A, UM-22B cells were treated with AR-12 (200 nmol/L), AG1478 (6 μmol/L), or erlotinib (7 μmol/L) or a combination of AR-12 plus AG1478 or erlotinib. MTT assay was carried out after 72 hours and the data analysis was done using GraphPad Prism Software. Experiments were conducted 3 times in 6 replicates (P = 0.003). B, athymic nude mice (n = 5) were inoculated with 1.5 × 106 PDK1 kinase-dead transfected HNSCC cells (PDKM) or vector-transfected control cells (VC) in the left and right flanks, respectively. After the formation of tumor nodules (7 days later), mice were treated with vehicle (saline) or cetuximab 3 times a week (0.8 mg i.p.) in conjunction with tumor volume determinations. C, athymic nude mice were inoculated with UMSCC1 cells. After 7 days, mice were randomized into 4 treatment groups shown (i) vehicle, (ii) AR-12, (iii) C225, and (iv) AR-12+C225. Tumors were measured every 3 days and tumor volume was calculated (length/2 × width2). Tumor volumes were graphed using Graph Pad prism Software.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure Legend - PDF File, 77KB.
- Supplementary Figure 1 - PDF file, 38KB, PDK1 siRNA prevents BK-induced growth in UMSCC-1 cells.
- Supplementary Figure 2 - PDF file, 76KB, Kinase-dead PDK1 decreases PGE2-induced growth increase.
- Supplementary Figure 3 - PDF file, 453KB, PDK1 contributes to HNSCC sensitivity to AR-12.
- Supplementary Figure 4 - PDF file, 106KB, AR-12 decreases cyclin D1 expression.
- Supplementary Figure 5 - PDF file, 289KB, Phosphorylated MAPK and AKT abrogated in C225-treated PDKM xenografts.
- Supplementary Figure 6 - PDF file, 201KB, Biochemical effect of PDK1 targeting strategies on Akt and p70S6K.
- Supplementary Table 1 - PDF file, 39KB, IC50 values for AR-12 in HNC and 1 normal cell line.
Volume 11, Issue 6
