Elevated expression of HSP90 and the antitumor effect of an HSP90 inhibitor via inactivation of the Akt/mTOR pathway in undifferentiated pleomorphic sarcoma

Abstract

Background: Undifferentiated pleomorphic sarcoma (UPS) is a heterogeneous tumor group, and little is known about molecular target therapy for UPS. Heat shock protein 90 (HSP90) is an expressed chaperone that refolds certain denatured proteins under stress conditions. One of these proteins is Akt. The disruption of Akt signaling plays an important role in tumor progression. The present study's purpose was to analyze the HSP90 expression, Akt/mTOR pathway activation and the correlation between HSP90 expression and its pathway activation in UPS.

Methods: The status of HSP90 and the profiles of the Akt/ mTOR pathway were assessed by immunohistochemistry in 79 samples of UPS, and these data were compared with clinicopathological and histopathological findings. The expressions of indicated proteins were assessed by Western blotting in five frozen samples. After treating UPS cells with the HSP90 inhibitor, we assessed the antitumor effect of the inhibitor.

Results: Immunohistochemically, phosphorylated Akt (p-Akt), p-mTOR, p-S6RP and p-4EBP were positive in 57.3, 51.9, 54.5 and 57.1% of the UPS samples, respectively. The expressions of those phosphorylated proteins were correlated with each other. HSP90 expression was elevated in 56.4% of the samples and was correlated with p-Akt, p-mTOR and p-S6RP. The immunohistochemical results were confirmed by Western blotting. The HSP90 inhibitor led to decreased viability and invasiveness of the cells and inactivated the AKT/mTOR pathway in vitro.

Conclusion: Elevated expression of HSP90 is a poor-prognosis factor and is involved in the activation of the Akt/mTOR pathway in UPS. HSP90 inhibition is a potential treatment option for UPS.

Fig. 1

Reclassification of “UPS-like” sarcomas to “pure” UPS. We excluded 32 tumors in the body cavity because the tumors could be a component of a sarcomatoid carcinoma or dedifferentiated liposarcoma (DDLS). Three tumors were reclassified as undifferentiated spindle cell sarcomas. Eight pleomorphic sarcomas with focal myxoid stroma were also excluded because the difference in the diagnosis between UPS with focal myxoid component and high-grade myxofibroarcoma (MFS) was ambiguous. Seven undifferentiated pleomorphic sarcomas with MDM2 amplification were excluded because their biological character is similar to that of DDLS. A FISH analysis showed an MDM2 red signal present in a cluster in a tumor cell nucleus (green: centromere of chromosome 12)

Fig. 2

Representative H&E stain (a) and immunohistochemical staining (b–g). Positive stains for p-Akt (b), p-mTOR (c), p-S6RP (d), and p-4EBP (e) in the Akt/mTOR pathway and p-MEK1/2 (f), and p-Erk1/2 (g) in the MAPK pathway. Decreased expression of PTEN in the tumor cells and immunostaining of endothelial cells (h). Immunohistochemical staining with different intensities for HSP90 (i). Immunohistochemically nuclear and/or cytoplasmic staining was judged as positive. Bar: 100 μm

Fig. 3

Kaplan-Meier survival curves for overall survival (OS) according to the results of the IHC analysis. Positivities for p-Akt, p-mTOR, p-S6RP, p-4EBP and HSP90 were correlated with OS by log-rank test. PTEN expression was not correlated with OS

Fig. 4

17-DMAG suppressed the growth of UPS cells and caused a decrease in the invasion of cells. (a) HSP90 inhibition decreased the viability in a dose- and time-dependent manner. (b) The Matrigel invasion assay showed that 17-DMAG caused a decrease in the invasion of both cell lines. Error bars = standard deviation.* p < 0.01, ** p < 0.05

Fig. 5

17-DMAG reduced the protein expression of the Akt/mTOR pathway. Cell lines were treated with 17-DMAG (0.3 μmol/L) for 6, 12 and 24 h. The 17-DMAG-induced changes in the activation status of the AKT/mTOR and MAPK pathways were evaluated by Western blotting. Decreased p-Akt, p-mTOR, p-S6RP, p-MEK1/2 and p-ERK1/2 expressions were observed in both cell lines. The HSP90 expressions showed no clear difference. The cell lines were also treated with or without 17-DMAG (0.3 μmol/L) diluted in DMSO for 24 h. DMSO had no influence on the status of the AKT/mTOR or MAPK pathways