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. 2007 Sep 17;97(6):785-91.
doi: 10.1038/sj.bjc.6603952.

PDK-1/AKT pathway as a novel therapeutic target in rhabdomyosarcoma cells using OSU-03012 compound

L Cen  1 , F-C Hsieh, H-J Lin, C-S Chen, S J Qualman, J Lin
Affiliations

PDK-1/AKT pathway as a novel therapeutic target in rhabdomyosarcoma cells using OSU-03012 compound

L Cen et al. Br J Cancer. .

Abstract

Rhabdomyosarcoma (RMS) is the most common paediatric soft-tissue sarcoma including two major subtypes, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). Increasing evidence suggests that oncogenesis of RMS involves multiple stages of signalling protein dysregulation which may include prolonged activation of serine/threonine kinases such as phosphoinositide-dependent kinase-1 (PDK-1) and AKT. To date, whether PDK-1/AKT pathway is activated in RMS is unknown. This study was to examine phosphorylation status of AKT and to evaluate a novel small molecular inhibitor, OSU-03012 targeting PDK-1 in RMS. We examined phosphorylation levels of AKT using ARMS and ERMS tissue microarray and immunohistochemistry staining. Our results showed phospho-AKT(Thr308) level is elevated 42 and 35% in ARMS and ERMS, respectively. Phospho-AKT(Ser473) level is also increased 43% in ARMS and 55% in ERMS. Furthermore, we showed that OSU-03012 inhibits cell viability and induces apoptosis in ARMS and ERMS cell lines (RH30, SMS-CTR), which express elevated phospho-AKT levels. Normal cells are much less sensitive to OSU-03012 and in which no detectable apoptosis was observed. This study showed, for the first time, that PDK-1/AKT pathway is activated in RMS and may play an important role in survival of RMS. PDK-1/AKT pathway may be an attractive therapeutic target for cancer intervention in RMS using OSU-03012.

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Figures

Figure 1
Figure 1
Expression of phosphorylated AKT in rhabdomyosarcoma (RMS) patient tissues and cell lines. (A) Immunohistochemical (IHC) staining of RMS tissue microarrays (TMAs) that contain both normal and cancer tissues using phospho-AKT (Thr308) and phospho-AKT (Ser473) antibodies. (B) The levels of phosphorylated AKT were examined using antibodies to threonine 308 and serine 473 in a series of RMS cell lines.
Figure 2
Figure 2
Effects of phosphoinositide-dependant kinase-1 (PDK-1) inhibitor OSU-03012 (OSU) on rhabdomyosarcoma (RMS) cell lines. (A) RH30 cells were treated for 8 h with 10 μM OSU-03012. OSU-03012 downregulates AKT phosphorylation at both threonine 308 and serine 473. Phosphorylation of p70S6K at threonine 229, one of the downstream targets of PDK-1, was also inhibited by OSU-03012. Poly(ADP-ribose) polymerase (PARP) cleavage was measured with anti-cleaved-PARP antibody by western blotting. (B) SMS-CTR cells were treated as above. OSU-03012 inhibits AKT phosphorylation at both threonine 308 and serine 473. Poly(ADP-ribose) polymerase (PARP) cleavage was measured with anti-cleaved-PARP antibody by western blotting. (C) RH30 cells were treated with 10 μM OSU-03012 for 4, 6 and 8 h respectively. AKT phosphorylation was inhibited in a time-dependent manner. (D) SMS-CTR cells were treated with 10 μM OSU-03012 for 2, 4, 6 and 8 h respectively. Time course inhibition of phosphorylated-AKT was shown by western blot.
Figure 3
Figure 3
Effects of OSU-03012 and LY294002 on cell viability (A–E). RH30 (A), SMS-CTR (B) and human fibroblast (HFF) (C) cells were exposed to OSU-03012 at the designated concentrations and cell viability was evaluated on days 1, 2 and 3, respectively. There was a dose-dependent decrease in cell viability upon treatment with increasing concentration of the inhibitor. Human fibroblast is much less sensitive to OSU-03012 compared to rhabdomyosarcoma cell lines. RH30 and SMS-CTR cells were treated with Ly294002 at the indicated concentrations and cell viability was evaluated on days 1, 2 and 3 respectively (D and E).
Figure 4
Figure 4
Effects of PDK-1 inhibitor OSU-03012 on apoptosis induction. (A and B) The cells were treated with 10 μM OSU-03012 for 24 h and fixed. Caspase-3 cleavage (shown in red) was evaluated by immunofluorescence staining with anti-cleaved-caspase-3 antibody. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining is shown in blue. Upon OSU-03012 treatment, RH30 and SMS-CTR cells showed an increased amount of caspase-3 cleavage (A). However, human fibroblast (HFF) and human skeletal muscle myoblast (HSMM) cells did not show any detectable caspase-3 cleavage after treatment (B).
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