HSP90 inhibition destabilizes Bcl-xL and induces apoptosis in MSP RAS cells 72

As Caldas-Lopes et al. have shown that Bcl-xL is a client of HSP90 and is therefore protected from proteasomal degradation [137], we were interested to see, whether HSP90 inhibition can destabilize Bcl-xL and may this affect the response to Cisplatin treatment. For this, we made use of the HSP90 inhibitors 17AAG as well as 17DMAG which disable the ATP binding site of HSP90 and thereby abolish HSP90 function [138]. MSP RAS cells were treated with HSP90 inhibitors for 24h followed by exposure to Cisplatin for 16h. Subsequently, the protein lysates were analyzed for Bcl-xL and induced apoptosis by western blot. We found that the inhibition of HSP90 in the absence of Cisplatin induced a decrease of Bcl-xL protein level as-sociated with active Caspase3 and cleaved PARP. The induction of apoptosis was enhanced in the presence of Cisplatin. The decrease of Akt protein level indicated the efficiency of the HSP90 inhibitors and is well detectable in the presence or absence of Cisplatin (Figure 5.19A). Next, it was tested whether the overexpression of Bcl-xL in 24⁺HMLE cells and MSP cells rescued the induction of apoptosis mediated by HSP90 inhibition. Indeed, neither in the absence nor in presence of Cisplatin apoptosis was induced in HMLE overexpressing Bcl-xL (Figure 5.19B).

It can be concluded that the inhibition of HSP90 results in a decrease of Bcl-xL level which in turn sensitizes MSP RAS cells to Cisplatin treatment. The high Bcl-xL levels in expression vector-containing HMLE cells rescued these cells from the induction of apoptosis mediated by HSP90 inhibition. This implicates that Bcl-xL plays a role in this apoptosis induction.

Figure 5.19: HSP90 inhibition sensitizes MSP RAS cells towards Cisplatin.

(A) 16h after seeding MSP RAS cells were treated with 5µM 17AAG or 17DMAG for 24h. Subsequently, cells were washed and exposed to 20µM Cisplatin for 16h. Thereafter, cells were harvested and lysates were analyzed for apoptosis and Bcl-xL by western blot. * indicates deamidated protein, # indicates unmodified protein.

n=3. (B) 16h after seeding 24+Bcl-xL, MSP Bcl-xL and MSP RAS were treated with 5µM 17AAG for 24h. Af-terwards, the cells were washed and treated with 40µM Cisplatin for 16h. Then, cells were harvested and ana-lyzed for apoptosis and Bcl-xL by western blot. n=1. Actin staining was used as loading control, while Akt stain-ing was used as positive control for the experiment. DMSO-treated cells were used as control. Arrows are indi-cating the intended staining.

Interestingly, the HSP90 inhibition with 17AAG and 17DMAG resulted in decreased Bcl-xL mRNA level, as well (Figure 5.20A). To examine, whether HSP90 directly causes a decrease in Bcl-xL mRNA level we performed a siRNA-mediated knock down of HSP90 and analyzed the Bcl-xL mRNA level 48h post-transfection with qRT-PCR. It was detectable that HSP90

knock down results in decreased Bcl-xL mRNA level, as well. However, this decrease was less prominent than that induced by 17AAG-mediated HSP90 inhibition (Figure 5.20B).

These data implicate that not only HSP90-mediated direct Bcl-xL protein stabilization is im-portant for the Bcl-xL level in MSP RAS cells. Rather, the stabilization of other HSP90 cli-ents affects Bcl-xL gene expression in these cells as well.

Figure 5.20: HSP90 inhibition decreases Bcl-xL mRNA level.

(A) 16h after seeding MSP RAS cells were treated with 5µM 17AAG or 17DMAG for 24h. Subsequently, cells were harvested and mRNA was isolated. mRNA levels of Bcl-xL were analyzed using qRT-PCR. DMSO-treated cells were used as control. (B) 48h after siRNA-mediated knock down of HSP90α and HSP90β, cells were har-vested, mRNA was isolated and mRNA levels of HSP90pan and Bcl-xL were analyzed by qRT-PCR. Non-transfected cells and scr siRNA were used as control. mRNA levels were normalized to 36B4. siRNA data show mean ±SD of n=3. Student´s t-test was performed for statistical analyses.

5.3.3 HSP90 inhibition reduces MSP RAS cell-derived tumors in mice

Previous results suggest the possibility to overcome the chemoresistance of MSP RAS cells by inhibiting HSP90 with 17DMAG. To this end, we were interested to examine whether MSP RAS cell-derived tumors in mice respond to 17DMAG under physiological conditions

as it was the case for MSP RAS cells in vitro. To test this, MSP RAS cells were injected into three week old (Nu/Nu) mice. In the phase of exponential tumor growth three weeks after in-jection, xenograft mice were treated with intraperitoneal injection of 17DMAG 5d per week until sacrifice. Tumor size was defined every day and plotted against time. This first experiment demonstrated that, 17DMAG treatment resulted in reduced tumor sizes compared to the control mice. This effect was reduced during the rest of the treatment (Figure 5.21).

These results support the notion that HSP90 inhibition constitutes a possibility to overcome chemoresistance in MSP RAS cells. Moreover, these findings suggest that this is even possi-ble under physiological conditions in mice. However, these first data have to be verified with a larger number of mice.

Figure 5.21: HSP90 inhibition via systemic 17DMAG treatment induces growth inhibition of MSP RAS cell-induced tumors in mice.

10⁶ MSP RAS cells were subcutaneously injected in (Nu/Nu) mice. Three weeks after injection, xenograft mice were treated with intraperitoneal injections of 10mg/kg/day 17DMAG (green line) or vehicle (5% glucose, or-ange line) 5d per week until sacrifice. During treatment, tumor sizes were monitored every day. (Preliminary da-ta; 4 tumors control group; 6 tumors 17DMAG group). Data show mean ±SD.