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. 2013 Dec;27(12):2397-403.
doi: 10.1038/leu.2013.150. Epub 2013 May 14.

Understanding the interplay between the proteasome pathway and autophagy in response to dual PI3K/mTOR inhibition in myeloma cells is essential for their effective clinical application

L I Aronson  1 , E L Davenport, F Mirabella, G J Morgan, F E Davies
Affiliations
Free PMC article

Understanding the interplay between the proteasome pathway and autophagy in response to dual PI3K/mTOR inhibition in myeloma cells is essential for their effective clinical application

L I Aronson et al. Leukemia. 2013 Dec.
Free PMC article
No abstract available

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Figures

Figure 1
Figure 1
Autophagy clears ubiquitinated proteins when proteasome stability is compromised by dual PI3K/mTOR inhibition. In order to demonstrate that PI-103 activated autophagy, (a) we stained cells with acridine orange and examined the development of acidic vesicles in response to PI-103, after 24 h treatment, by confocal microscopy at 63x magnification (top panels). Acridine orange fluoresces red in an acidic milieu such as autophagosomes but fluoresces green when bound to DNA. Treatment with the autophagy inhibitor, Bfa, for the final 1 h of treatment reduced the formation of these vesicles (bottom panels). Scale bars represent 10 μm. (b) Results obtained by confocal microscopy were confirmed in triplicate by flow cytometry in MM.1S and primary patient samples. Treatment with increasing concentrations of PI-103 for 24 h resulted in an increase in the ratio of red:green fluorescence (1st and 3rd panels), while 1h treatment with 25 nmol/l Bfa blocked this increase at all tested concentrations (2nd and 3rd panels). The same pattern was observed in patient cells (bottom panel). (c) Further evidence for autophagy induction is shown by western blotting in MM.1S for the autophagosome marker, LC3II, and p62 over a 24 h timecourse with PI-103. (d) Activation of autophagy by PI-103 was accompanied by inhibition of the chymotrypsin-like catalytic activity of the proteasome by 24 h as shown in MM.1S and H929. The decrease observed in U266 was not significant. **P<0.01. Results represent mean of three separate experiments. Bortezomib was used as a positive control. (e) Inhibition of the proteasome can lead to an increase in the UPR in order to cope with excess proteins in the cell. Treatment of MM.1S, H929 and U266 with PI-103 for 24 h lead to an increase in UPR markers, XBP1s, CHOP and ATF4, as assessed by reverse transcription-PCR (RT-PCR). Data were normalized using actin. Representative plots are shown. (f) Competitive binding assay with a fluorescently labeled proteasome activity probe (Me4BodipyFL-Ahx3Leu3VS) showed that proteasome inhibition by PI-103 was not due to inhibition of the catalytic sites, as no change was observed in any of the proteasome β subunits in response to PI-103 over 24 h. Bortezomib was used as a positive control and actin as a loading control. (g) To demonstrate proteasome inhibition visually, we performed western blotting for ubiquitin in both the soluble and insoluble cell fractions. In MM.1S, H929 and U266, ubiquitin decreased over 24 h in response to PI-103. As can be seen in the insoluble fractions, ubiquitin was not accumulating as insoluble aggregates either. Blotting for p62 and LC3 showed a decrease and increase, respectively, in response to PI-103 indicative of autophagy activation. (h) Relative expression of proteasome and autophagy genes was examined in MM.1S using Affymetrix Human Gene ST 2.0 arrays at 6 and 12 h after PI-103 treatment. The indicated genes had at least a 1.5-fold change over the timecourse. Asterisks denote components of the proteasome 19S lid and base. Drug concentrations are as follows unless otherwise stated. PI-103: MM.1S and H929, 2 μmol/l; U266, 5.5 μmol/l. Bfa: MM.1S and H929, 100 nmol/l; U266, 200 nmol/l. Bortezomib: MM.1S, H929 and U266, 8 nmol/l.
Figure 1
Figure 1
Autophagy clears ubiquitinated proteins when proteasome stability is compromised by dual PI3K/mTOR inhibition. In order to demonstrate that PI-103 activated autophagy, (a) we stained cells with acridine orange and examined the development of acidic vesicles in response to PI-103, after 24 h treatment, by confocal microscopy at 63x magnification (top panels). Acridine orange fluoresces red in an acidic milieu such as autophagosomes but fluoresces green when bound to DNA. Treatment with the autophagy inhibitor, Bfa, for the final 1 h of treatment reduced the formation of these vesicles (bottom panels). Scale bars represent 10 μm. (b) Results obtained by confocal microscopy were confirmed in triplicate by flow cytometry in MM.1S and primary patient samples. Treatment with increasing concentrations of PI-103 for 24 h resulted in an increase in the ratio of red:green fluorescence (1st and 3rd panels), while 1h treatment with 25 nmol/l Bfa blocked this increase at all tested concentrations (2nd and 3rd panels). The same pattern was observed in patient cells (bottom panel). (c) Further evidence for autophagy induction is shown by western blotting in MM.1S for the autophagosome marker, LC3II, and p62 over a 24 h timecourse with PI-103. (d) Activation of autophagy by PI-103 was accompanied by inhibition of the chymotrypsin-like catalytic activity of the proteasome by 24 h as shown in MM.1S and H929. The decrease observed in U266 was not significant. **P<0.01. Results represent mean of three separate experiments. Bortezomib was used as a positive control. (e) Inhibition of the proteasome can lead to an increase in the UPR in order to cope with excess proteins in the cell. Treatment of MM.1S, H929 and U266 with PI-103 for 24 h lead to an increase in UPR markers, XBP1s, CHOP and ATF4, as assessed by reverse transcription-PCR (RT-PCR). Data were normalized using actin. Representative plots are shown. (f) Competitive binding assay with a fluorescently labeled proteasome activity probe (Me4BodipyFL-Ahx3Leu3VS) showed that proteasome inhibition by PI-103 was not due to inhibition of the catalytic sites, as no change was observed in any of the proteasome β subunits in response to PI-103 over 24 h. Bortezomib was used as a positive control and actin as a loading control. (g) To demonstrate proteasome inhibition visually, we performed western blotting for ubiquitin in both the soluble and insoluble cell fractions. In MM.1S, H929 and U266, ubiquitin decreased over 24 h in response to PI-103. As can be seen in the insoluble fractions, ubiquitin was not accumulating as insoluble aggregates either. Blotting for p62 and LC3 showed a decrease and increase, respectively, in response to PI-103 indicative of autophagy activation. (h) Relative expression of proteasome and autophagy genes was examined in MM.1S using Affymetrix Human Gene ST 2.0 arrays at 6 and 12 h after PI-103 treatment. The indicated genes had at least a 1.5-fold change over the timecourse. Asterisks denote components of the proteasome 19S lid and base. Drug concentrations are as follows unless otherwise stated. PI-103: MM.1S and H929, 2 μmol/l; U266, 5.5 μmol/l. Bfa: MM.1S and H929, 100 nmol/l; U266, 200 nmol/l. Bortezomib: MM.1S, H929 and U266, 8 nmol/l.
Figure 2
Figure 2
Dual PI3K/mTOR inhibition, combined with autophagy inhibition, enhances apoptosis of myeloma cells. Given that PI-103 induces autophagy in myeloma cells, we were interested to understand the effect on cells when autophagy was blocked. (a) The combination ofPI-103, with a constant subtoxic concentration of Bfa, over 24 h effectively inhibited the proliferation of MM.1S and H929 (left panels) at all PI-103 concentrations tested, as determined by triplicate MTT assays. **P<0.01. The combination was no more effective in U266 than PI-103 alone (right panel). (b) Initiation of apoptosis in MM.1S (left) and U266 (right) was confirmed by trypan blue exclusion and Annexin V/PI (propidium iodide) staining over a 24 h period with PI-103 and Bfa. Results represent the average of three independent experiments. (c) Specificity of the observed effect with PI-103 and Bfa was demonstrated by triplicate MTT assay by treating cells for 24 h with a second PI3K/mTOR inhibitor, BEZ235, in combination with another autophagy inhibitor, CHQ. When CHQ was applied at a constant subtoxic concentration, it significantly inhibited proliferation of MM.1S, but not U266, at all tested concentrations of BEZ235. (d) This was confirmed, in triplicate, by Annexin V/PI staining, indicating that apoptosis is enhanced by combination treatment in MM.1S but not U266. (e) In order to delineate whether it was the inhibition of PI3K or mTOR that was more important for the observed effect, we performed triplicate MTT assays with the mTOR inhibitor, Rapamycin, in combination with Bfa for 24 h. The combination significantly enhanced apoptosis at most of the tested Rapamycin concentrations in MM.1S but not in H929 or U266. (f) Left, PI-103 had minimal effect on patient-derived BMSCs (BMSC 1-3) seeded at two different densities (5 × 104 (5) and 10 × 104 (10) cells per well) as assessed by 24 h Wst1 assays. Right, Using conditioned medium from patient BMSCs, 24 h MTT assays were performed to determine the efficacy of the combination in MM.1S in the presence of bone marrow cytokines. Representative results from one of the three experiments are shown. CM, conditioned medium; normal, normal growth medium. (g) The chymotrypsin-like activity of the proteasome was determined in triplicate in MM.1S and U266 following 6 and 24 h treatment with PI-103, Bfa or both. Bortezomib (Bort) was used as a positive control. *P<0.05; **P<0.01. (h) Detection of UPR markers, XBP1s, CHOP and ATF4, by RT-PCR in MM.1S and U266 following 6 and 24 h treatment with PI-103, Bfa or both. Data were normalized using actin. Representative plots are shown.(i) Western blot analysis was carried out at 24 h in MM.1S (left) and U266 (right) treated with PI-103, Bfa or both. Drug concentrations were as follows. PI-103: MM.1S, 2 μmol/l; U266, 5.5 μmol/l. Bfa: MM.1S and H929, 100 nmol/l; U266, 200 nmol/l. BEZ235: MM.1S, 800 nmol/l; U266, 25 μmol/l. CHQ: MM.1S and U266, 500 nmol/l. Bortezomib was used at 8 nmol/l for both cell lines.
Figure 2
Figure 2
Dual PI3K/mTOR inhibition, combined with autophagy inhibition, enhances apoptosis of myeloma cells. Given that PI-103 induces autophagy in myeloma cells, we were interested to understand the effect on cells when autophagy was blocked. (a) The combination ofPI-103, with a constant subtoxic concentration of Bfa, over 24 h effectively inhibited the proliferation of MM.1S and H929 (left panels) at all PI-103 concentrations tested, as determined by triplicate MTT assays. **P<0.01. The combination was no more effective in U266 than PI-103 alone (right panel). (b) Initiation of apoptosis in MM.1S (left) and U266 (right) was confirmed by trypan blue exclusion and Annexin V/PI (propidium iodide) staining over a 24 h period with PI-103 and Bfa. Results represent the average of three independent experiments. (c) Specificity of the observed effect with PI-103 and Bfa was demonstrated by triplicate MTT assay by treating cells for 24 h with a second PI3K/mTOR inhibitor, BEZ235, in combination with another autophagy inhibitor, CHQ. When CHQ was applied at a constant subtoxic concentration, it significantly inhibited proliferation of MM.1S, but not U266, at all tested concentrations of BEZ235. (d) This was confirmed, in triplicate, by Annexin V/PI staining, indicating that apoptosis is enhanced by combination treatment in MM.1S but not U266. (e) In order to delineate whether it was the inhibition of PI3K or mTOR that was more important for the observed effect, we performed triplicate MTT assays with the mTOR inhibitor, Rapamycin, in combination with Bfa for 24 h. The combination significantly enhanced apoptosis at most of the tested Rapamycin concentrations in MM.1S but not in H929 or U266. (f) Left, PI-103 had minimal effect on patient-derived BMSCs (BMSC 1-3) seeded at two different densities (5 × 104 (5) and 10 × 104 (10) cells per well) as assessed by 24 h Wst1 assays. Right, Using conditioned medium from patient BMSCs, 24 h MTT assays were performed to determine the efficacy of the combination in MM.1S in the presence of bone marrow cytokines. Representative results from one of the three experiments are shown. CM, conditioned medium; normal, normal growth medium. (g) The chymotrypsin-like activity of the proteasome was determined in triplicate in MM.1S and U266 following 6 and 24 h treatment with PI-103, Bfa or both. Bortezomib (Bort) was used as a positive control. *P<0.05; **P<0.01. (h) Detection of UPR markers, XBP1s, CHOP and ATF4, by RT-PCR in MM.1S and U266 following 6 and 24 h treatment with PI-103, Bfa or both. Data were normalized using actin. Representative plots are shown.(i) Western blot analysis was carried out at 24 h in MM.1S (left) and U266 (right) treated with PI-103, Bfa or both. Drug concentrations were as follows. PI-103: MM.1S, 2 μmol/l; U266, 5.5 μmol/l. Bfa: MM.1S and H929, 100 nmol/l; U266, 200 nmol/l. BEZ235: MM.1S, 800 nmol/l; U266, 25 μmol/l. CHQ: MM.1S and U266, 500 nmol/l. Bortezomib was used at 8 nmol/l for both cell lines.
Figure 2
Figure 2
Dual PI3K/mTOR inhibition, combined with autophagy inhibition, enhances apoptosis of myeloma cells. Given that PI-103 induces autophagy in myeloma cells, we were interested to understand the effect on cells when autophagy was blocked. (a) The combination ofPI-103, with a constant subtoxic concentration of Bfa, over 24 h effectively inhibited the proliferation of MM.1S and H929 (left panels) at all PI-103 concentrations tested, as determined by triplicate MTT assays. **P<0.01. The combination was no more effective in U266 than PI-103 alone (right panel). (b) Initiation of apoptosis in MM.1S (left) and U266 (right) was confirmed by trypan blue exclusion and Annexin V/PI (propidium iodide) staining over a 24 h period with PI-103 and Bfa. Results represent the average of three independent experiments. (c) Specificity of the observed effect with PI-103 and Bfa was demonstrated by triplicate MTT assay by treating cells for 24 h with a second PI3K/mTOR inhibitor, BEZ235, in combination with another autophagy inhibitor, CHQ. When CHQ was applied at a constant subtoxic concentration, it significantly inhibited proliferation of MM.1S, but not U266, at all tested concentrations of BEZ235. (d) This was confirmed, in triplicate, by Annexin V/PI staining, indicating that apoptosis is enhanced by combination treatment in MM.1S but not U266. (e) In order to delineate whether it was the inhibition of PI3K or mTOR that was more important for the observed effect, we performed triplicate MTT assays with the mTOR inhibitor, Rapamycin, in combination with Bfa for 24 h. The combination significantly enhanced apoptosis at most of the tested Rapamycin concentrations in MM.1S but not in H929 or U266. (f) Left, PI-103 had minimal effect on patient-derived BMSCs (BMSC 1-3) seeded at two different densities (5 × 104 (5) and 10 × 104 (10) cells per well) as assessed by 24 h Wst1 assays. Right, Using conditioned medium from patient BMSCs, 24 h MTT assays were performed to determine the efficacy of the combination in MM.1S in the presence of bone marrow cytokines. Representative results from one of the three experiments are shown. CM, conditioned medium; normal, normal growth medium. (g) The chymotrypsin-like activity of the proteasome was determined in triplicate in MM.1S and U266 following 6 and 24 h treatment with PI-103, Bfa or both. Bortezomib (Bort) was used as a positive control. *P<0.05; **P<0.01. (h) Detection of UPR markers, XBP1s, CHOP and ATF4, by RT-PCR in MM.1S and U266 following 6 and 24 h treatment with PI-103, Bfa or both. Data were normalized using actin. Representative plots are shown.(i) Western blot analysis was carried out at 24 h in MM.1S (left) and U266 (right) treated with PI-103, Bfa or both. Drug concentrations were as follows. PI-103: MM.1S, 2 μmol/l; U266, 5.5 μmol/l. Bfa: MM.1S and H929, 100 nmol/l; U266, 200 nmol/l. BEZ235: MM.1S, 800 nmol/l; U266, 25 μmol/l. CHQ: MM.1S and U266, 500 nmol/l. Bortezomib was used at 8 nmol/l for both cell lines.
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References

    1. Aronson LI, Davies FE. DangER: protein ovERload. Targeting protein degradation to treat myeloma. Haematologica. 2012;97:1119–1130. - PMC - PubMed
    1. Davenport EL, Moore HE, Dunlop AS, Sharp SY, Workman P, Morgan GJ, et al. Heat shock protein inhibition is associated with activation of the unfolded protein response pathway in myeloma plasma cells. Blood. 2007;110:2641–2649. - PubMed
    1. Hoang B, Benavides A, Shi Y, Frost P, Lichtenstein A. Effect of autophagy on multiple myeloma cell viability. Mol Cancer Ther. 2009;8:1974–1984. - PubMed
    1. Iwata A, Riley BE, Johnston JA, Kopito RR. HDAC6 and microtubules are required for autophagic degradation of aggregated huntingtin. J Biol Chem. 2005;280:40282–40292. - PubMed
    1. Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, et al. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature. 2006;441:885–889. - PubMed

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