Apoptosis induction in ES cells after inhibitor combinations
We treated p53wt WE-68 and p53−/− A673 cells with AUY922, as well as with VE821 to study p53-dependent effects. In comparison, we also applied the ATM inhibitor (ATMi) KU55933 to study the role of different DDR pathways upon HSP90 inhibition. As readouts, we measured the loss of the mitochondrial transmembrane potential (ΔΨM), a marker for apoptosis [19, 17, 20], and cell death by flow cytometry. We combined AUY922 with either VE821 (short AUY-VE) or KU55933 (short AUY-KU). Low nanomolar concentrations of AUY922 induced ΔΨM loss and cell death in both ES cell lines (Fig. 1A-D). Yet combinations with either VE821 or KU55933 enhanced the cytotoxicity of AUY922, with a stronger effect of AUY-VE (Fig. 1A-D). The ΔΨM decay reached 90% in WE-68 cells, but only 64% in A673 cells after AUY-VE (compare Fig. 1A and C), suggesting a p53-dependent induction of mitochondrial-mediated apoptosis in p53wt cells.
Amplification of AUY922-mediated cellular damages by AUY-VE but not by AUY-KU
Since HSP90i are known to induce DNA damage [21], we analyzed the expression level of key DDR molecules, together with DNA damage markers by Western blotting. AUY922 and AUY-VE strongly reduced the expression of ATR and CHK1 and mildly reduced that of ATM, while they did not affect CHK2 protein levels in both ES cell lines (Fig. 1E-G). Interestingly, BRCA1 expression showed cell line specific responses to the treatments: it was decreased in A673 cells and increased in WE-68 cells (particularly more obvious at high concentrations). AUY-KU, however, increased the abundance of all studied DDR proteins in WE-68 cells and of ATM and CHK1 in A673 cells.
In addition, AUY922 and AUY-VE induced signs of apoptosis and DNA damage. High concentrations of AUY922 induced the expression of p53, phosphorylation of p53 (p-p53) and of H2AX (γH2AX), both signs of active DDR [22, 23], as well as the cleavage of PARP1 (cl-PARP1) and caspase-3 (cl-caspase3) in WE-68 cells, indicative of ongoing apoptosis [17] (Fig. 1E). AUY-VE treatments strongly increased the level of γH2AX, cl-caspase3 and cl-PARP1, p53 and p-p53. In contrast, AUY-KU abolished their presence, suggesting a protective function of KU55933 (Fig. 1E). In A673 cells, AUY922 also induced cl-caspase3 and cl-PARP1 but to a lesser extent (comparing Fig. 1E and 1F). However, the level of γH2AX was decreased at high concentrations of AUY922, likely due to a strong depletion of DDR proteins, i.e., ATM and ATR in A673 cells that impaired DNA damage recognition [23]. AUY-VE and AUY-KU enhanced the level of γH2AX and cl-PARP1, but not of cl-caspase3, whereas the AUY-VE effects were stronger and more persistent (Fig. 1F).
Increased treatment effectiveness of AUY-VE in p53 wild-type ES cells
This led us to study whether the effects of the drug treatment are reflected in cell proliferation. A crystal violet-based assay showed that AUY922 decreased cell proliferation in the ES cell lines in a concentration- and time-dependent fashion. After 24 h, cell densities were already significantly reduced by AUY922, with stronger effects being observed in WE-68 (61%; p < 0.001) than in A673 cells (23%; p < 0.05) (Fig. 2A,B and Fig. S1A,B). The effects were even more pronounced after 48 h. Cell densities of WE-68 cells went close to zero after 72 h treatment with 30–45 nM AUY922 (p < 0.001), whereas cell densities of A673 cells after 72 h were just reduced about 20% compared to their initial control value at 24 h and thereafter remained unchanged (Fig. 2A,B). Remarkably, AUY-VE enhanced the effectiveness on WE-68 cells proliferation after 48 and 72 h co-treatment. To our surprise, we did not find differences in cell densities of A673 cells at any time point after AUY-VE or AUY-KU compared to AUY922 alone (Fig. 2B). We speculated that ES cells underwent cell cycle arrest due to the treatments. However, flow cytometry did not detect any prominent alterations of cell cycle profiles (Fig. S1C-G). The weaker effects on cell proliferation (density) observed in A673 cells were likely the result of lower apoptosis level because of p53 deficiency.
AUY-VE is suitable to treat other types of cancer beyond ES
To analyze if our observations could be generalized or are limited to ES cells, we used isogenic p53wt and p53−/− HCT116 cells and analyzed them by Western blotting (Fig. 2A). AUY922 decreased the expression of ATR, ATM and BRAC1 in both cell lines. VE821 and AUY-VE had no effect on ATM stability but further reduced the levels of BRCA1 and ATR. HCT116 p53wt cells had more cl-caspase3 after AUY922 treatment, which was further increased after AUY-VE treatment, whereas p53−/− cells had less cl-caspase3, despite more DNA damage, judged by γH2AX. Similarly, the level of cl-PARP1 was increased more in p53wt than in p53−/− cells after AUY922 and AUY-VE treatment. Overall, we found similar protein alterations in both studied cancer cell types (compare Fig. 1E,F and Fig. 2A) by AUY and AUY-VE, with stronger effects in ES cells. Furthermore, we performed a BALB/c cell transformation assay (BALB-CTA) [18] to study the therapeutic potential of AUY-VE (Fig. 2C,D and Fig. S2B). Malignant transformation of BALB/c cells was induced via consecutive MCA and TPA treatments. AUY922 (30–45 nM) reduced the number of type-III foci (Fig. 2C,D). AUY-VE led to a further reduction even at low concentrations (15 or 45 nM AUY922, p < 0.05; Fig. 2D). Thus, AUY-VE showed therapeutic potential beyond ES cells.
Activation of p53 target gene transcription in WE-68 cells after AUY-VE treatment
To gain further molecular understanding, we analyzed mRNA expression level by qPCR (Fig. 3). In WE-68 cells, we saw upregulated levels of the pro-apoptotic BAK1, BAX (p < 0.01) and BCL2L11 (BIM) (p < 0.05) after AUY922 treatment, with the p53 target gene BAX [24] showing the strongest effects. AUY-VE further increased, whereas AUY-KU decreased their mRNA levels (Fig. 3A). In A673 cells, we found no change of BAK1 and BAX, but an increase of BCL2L11 expression after AUY922 (p < 0.01; p < 0.05 to WE-68) treatment, which was not affected by AUY-VE or AUY-KU. Among anti-apoptotic BCL2 family members, BCL2L1 (BCL-Xl) and MCL1 mRNAs remained unchanged in both ES cell lines (Fig. 3B). BCL2 was slightly increased after AUY922 (p < 0.001) in WE-68 but not in A673 cells, which were further enhanced by AUY-KU (p < 0.05 to A673). Since we discovered recently an important function of AIF in p53 null ES cells [19], we also determined AIFM1 (AIF) mRNA expression after AUY922 treatments (Fig. S2C). However, we found only a mildly elevated AIFM1 (p < 0.05 for A673) in both ES cell lines, which was however not affected by AUY-VE or AUY-KU. Thus, AIF seemed to be not involved in apoptosis induction after AUY-VE treatment.
Next, we analyzed more p53 target genes and found that AUY922 increased the mRNA expression of CDKN1A (p21) and pro-apoptotic BBC3 (PUMA) [24] (p < 0.05 for both), which were both greatly further enhanced by AUY-VE in WE-68 cells (Fig. 3C). In contrast, AUY-KU did not change CDKN1A and BBC3 mRNAs levels. The mRNA of pro-apoptotic PMAIP1 (NOXA) [24] was increased after AUY922 treatment (p < 0.01 in both ES cell lines) and remained unchanged after AUY-KU and AUY-VE. Further, we found a small increase of TP53 (p < 0.01) after AUY922 in WE-68 cells, which was not affected by AUY-VE or AUY-KU (Fig. S2D). In contrast, MDM2 levels were significantly increased after AUY922 (p < 0.01), further enhanced by AUY-VE, but decreased by AUY-KU (Fig. S2D), similar to the pattern of pro-apoptotic p53 target genes (Fig. 3A,C).
Since the treatments had affected the expression of DDR proteins, we also investigated the mRNA levels of ATR, ATM, CHEK1 and CHEK2 and found that ATM and CHEK1 mRNAs were not impaired by the treatments. The expression of ATR (p < 0.05 for A673; p < 0.01 for WE-68) and CHEK2 (p < 0.01 for A673; p < 0.05 for WE-68) was increased by AUY922 but not affected by AUY-VE or AUY-KU (Fig. 3D,E). These results suggest that the decay of DDR proteins (see Fig. 1E,F) was not the result of altered gene expression, but rather of reduced protein stabilities. Taken together, upregulation of pro-apoptotic p53 target gene expression by AUY922 and further enhanced by AUY-VE can explain the strong induction of apoptosis in p53wt WE-68 cells (Fig. 1A-D).
Pro-apoptotic ER stress occurs in A673 cells after AUY-VE treatment
In both ES cell lines, BIM and DR5 accumulated in response to AUY922 (Fig. 4A-C), which reflect endoplasmic reticulum (ER) stress [25, 26]. Western blot analysis revealed that AUY-VE had no additional effect on DR5, but further increased BIM levels in A673 cells. AUY-KU had no additional effect on BIM, but decreased the expression of DR5 in both ES cell lines (Fig. 4A-C). Flow cytometry showed that reactive oxygen species (ROS), another sign of ER stress [27], were decreased in WE-68 cells after AUY922 treatment (Fig. S2E). In contrast, they accumulated in A673 cells and reached a plateau at 30–45 nM. The amount of ROS, however, was not affected by AUY-VE or AUY-KU in the ES cell lines.
To study the autophagy response as a result of ER stress, we analyzed LC3B, p62 and LAMP1 expressions [28, 29] (Fig. 4D-F). P62 decreased in both ES cell lines in response to AUY922 and AUY-VE. AUY-KU increased the p62 level in WE-68 cells, but had no additional effect in A673 cells compared to AUY922. LAMP1 was decreased after AUY922, which however was stabilized by AUY-VE in both ES cell lines. The total amount of LC3B was only mildly affected by AUY922 or AUY-VE in WE-68 cells, whereas more LC3B-I converted to LC3B-II after both treatments, indicating ongoing autophagy [28]. In A673 cells, the total LC3B level was increased by AUY-VE, indicative of stronger autophagy in these cells, whereas none of the treatments changed the LC3B-II/-I ratio. In sum, AUY922 induced in both ES cell lines pro-apoptotic ER stress and autophagy, and AUY-VE particularly enhanced them in A673 cells. AUY-KU showed again opposite effects to AUY-VE.
Proteome analysis of A673 cells validates ER stress induction after AUY-VE
Finally, we performed quantitative mass spectrometry analysis of the whole A673 cell proteome to validate our findings (Fig. 5, Figs. S3,4; for individual protein changes refer to File S1). AUY922 and AUY-VE induced the strongest proteome alterations (Fig. S3A-C and File S1), which was reflected by PCA analysis (Fig. S3D). Ingenuity pathway analysis (IPA) of the data sets (cutoff: q < 0.05) (Fig. 5A-D and Fig. S4A,) showed that both AUY922 and AUY-VE decreased DNA maintenance pathways alongside growth factor signaling and cytoskeletal organization, but activated the chaperone regulator BAG2 and PI3K/AKT signaling (Fig. 5A), although corresponding proteins decreased (File S1). In addition, all treatments reduced cell viability and proliferation of A673 cells and activated cell death and apoptosis (Fig. 5B), with the strongest effect by AUY-VE. These pathway alterations were regulated by proteins involved in cell cycle regulation and proliferation, inflammation, DNA damage recognition, cancer progression and stress response [30–35] (Fig. 5C). IPA further predicts strong mitochondrial dysfunctions, oxidative stress and changes of fatty acid (FA) metabolism after AUY922 and VE821 single-treatments (Fig. S4A,B) and most prominently after AUY-VE (Fig. 5D).
Moreover, we found an overlap of 208 proteins among the differentially expressed proteins (DEPs; cutoff: q < 0.05) of all three treatments, which were altered in the same direction (Fig. 5E and File S1). IPA showed that they are involved in apoptosis and cell proliferation, FA and cholesterol metabolism (Fig. S4C,D). In parallel, we found 630 DEPs (cutoff: q < 0.05) which were unique for AUY-VE (Fig. 5E and File S1) and IPA showed that they were involved in reduced cell survival, cytoskeletal organization, DNA maintenance, increased cell death, oxidative stress and mitochondrial dysfunctions (Fig. S4E,F). These findings indicate that AUY922, VE821 and their combination share common molecular targets, which mediate the synergisms between both compounds. In correlation with our initial experiment (Fig. 1), AUY-VE further promoted cell death in A673 cells by increasing mitochondrial dysfunctions and DNA damages.