Antimyeloma activity of heat shock protein-90 inhibition - PubMed (original) (raw)

. 2006 Feb 1;107(3):1092-100.

doi: 10.1182/blood-2005-03-1158. Epub 2005 Oct 18.

Nicholas S Mitsiades, Ciaran J McMullan, Vassiliki Poulaki, Andrew L Kung, Faith E Davies, Gareth Morgan, Masaharu Akiyama, Reshma Shringarpure, Nikhil C Munshi, Paul G Richardson, Teru Hideshima, Dharminder Chauhan, Xuesong Gu, Charles Bailey, Marie Joseph, Towia A Libermann, Neal S Rosen, Kenneth C Anderson

Affiliations

Antimyeloma activity of heat shock protein-90 inhibition

Constantine S Mitsiades et al. Blood. 2006.

Abstract

We show that multiple myeloma (MM), the second most commonly diagnosed hematologic malignancy, is responsive to hsp90 inhibitors in vitro and in a clinically relevant orthotopic in vivo model, even though this disease does not depend on HER2/neu, bcr/abl, androgen or estrogen receptors, or other hsp90 chaperoning clients which are hallmarks of tumor types traditionally viewed as attractive clinical settings for use of hsp90 inhibitors, such as the geldanamycin analog 17-AAG. This class of agents simultaneously suppresses in MM cells the expression and/or function of multiple levels of insulin-like growth factor receptor (IGF-1R) and interleukin-6 receptor (IL-6R) signaling (eg, IKK/NF-kappaB, PI-3K/Akt, and Raf/MAPK) and downstream effectors (eg, proteasome, telomerase, and HIF-1alpha activities). These pleiotropic proapoptotic effects allow hsp90 inhibitors to abrogate bone marrow stromal cell-derived protection on MM tumor cells, and sensitize them to other anticancer agents, including cytotoxic chemotherapy and the proteasome inhibitor bortezomib. These results indicate that hsp90 can be targeted therapeutically in neoplasias that may not express or depend on molecules previously considered to be the main hsp90 client proteins. This suggests a more general role for hsp90 in chaperoning tumor- or tissue-type-specific constellations of client proteins with critical involvement in proliferative and antiapoptotic cellular responses, and paves the way for more extensive future therapeutic applications of hsp90 inhibition in diverse neoplasias, including MM.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

In vitro anti-MM activity of hsp90 inhibitors. (A) In vitro anti-MM activity of geldanamycin against both drug-sensitive and drug-resistant MM cell lines, including the Dex-resistant MM-1R and the doxorubicin-resistant RPMI-8226/Dox40 cells. (B) Comparative in vitro activity of geldanamycin and its analogs against MM-1S myeloma cells. (C) In vitro activity of the geldanamycin analog 17-AAG (NSC 330507) against primary MM tumor cells isolated from patients resistant to conventional or investigational therapies (including conventional or high-dose chemotherapy, thalidomide, IMiDs, or the proteasome inhibitor PS-341). For each sample, red bars represent the percent survival (mean ± SD) of drug-treated cells; blue bars, the survival of their respective controls. (D) Dose-response matrix of human MM cell lines (as well as cells from other B-cell malignancies, such as IM-9 and ARH-77) to 17-AAG in vitro (0-5 μM for 72 hours). The survival of 17-AAG-treated cells (expressed as percent of the vehicle-treated control) is visualized in color format according to their values on a linear scale (0-100%).

Figure 2.

Figure 2.

In vivo anti-myeloma activity of hsp90 inhibition. (A) Representative results of whole-body fluorescence imaging of SCID/NOD mice injected intravenously with 5 × 106 RPMI-8226/S-GFP+ human MM cells and receiving either 17-AAG treatment or vehicle. Imaging results obtained 4 weeks after intravenous injection of GFP+ MM cells indicate extensive diffuse GFP+ bone lesions (arrows) in the spine, skull, lower extremities, and pelvis, as well as subcutaneous GFP+ lesions in the control cohort, in marked contrast to the absence of MM lesions in 17-AAG-treated mice. (B) Kaplan-Meier survival curve of 17-AAG-treated vs. control SCID/NOD mice injected intravenously with RPMI-8226/S-GFP+ cells. 17-AAG treatment significantly prolonged median overall survival of mice (> 250 days, with 14/20 of 17-AAG-treated mice surviving at the last interim analysis, vs 29 days median overall survival for control mice, with 0/20 mice surviving) (P < .001, log-rank test).

Figure 3.

Figure 3.

Functional sequelae of hsp90 inhibition. (A) Immunoblotting analyses of MM-1S cells treated with 17-AAG (500 nM for 0-24 hours) confirms that hsp90 inhibition suppresses the intracellular levels of multiple downstream effectors of the IGF-1R and IL-6R pathways, including the Raf-1 and IKK-α kinases (coupled with decreased constitutive phosphorylation of MEK1/2), the antiapoptotic proteins FLIP, XIAP, A1/bfl-1, cIAP2, and the pro-osteoclastogenic stimulator RANKL. In contrast, hsp90 inhibition by 17-AAG does not confer a significant effect on the intracellular levels of MEK1/2, for example. (B) Telomerase activity assay (by densitometric evaluation of the telomeric repeat amplification protocol (TRAP) method indicates that 17-AAG (500 nM, 0-24 hours) suppresses both constitutive and IGF-1 (200 ng/mL)-induced activation of the catalytic subunit of telomerase (hTERT). (C) 17-AAG treatment (750 nM for 0-36 hours) suppresses the NF-κB activity of MM-1S, as evidenced by NF-κB DNA binding enzyme-linked immunosorbent assay (ELISA). (D) 17-AAG treatment (750 nM for 0-36 hours) suppresses the activity of the 20S proteasome, as evidenced by 20S chymotryptic activity assay. (B-D) Error bars indicate SD.

Figure 4.

Figure 4.

Hsp90 inhibition targets IGF-1R and IL-6R signaling. Flow cytometric analysis of 17-AAG-treated (750 nM for 24 hours) MM-1S cells indicates complete abrogation of cell surface expression of IGF-1R (CD221) and suppression of surface IL-6R (CD126). The lack of significant suppression of cell surface expression of CD40, another receptor implicated in regulation of MM cell pathophysiology, supports the notion of specificity of the role of hsp90 on surface expression and function of certain growth/survival receptors. Filled curves correspond to staining with anti-IGF-1R, anti-IL-6R, or anti-CD40 antibody; open curves, staining with the respective isotype control antibody.

Figure 5.

Figure 5.

In vivo molecular profiling of hsp90 inhibition. MM-1S-GFP+/Luc+ cells injected intravenously in SCID/NOD mice led to development of diffuse bone lesions. Mice were randomly assigned to receive either a single dose of 17-AAG (50 mg/kg intraperitoneally) or vehicle; 24 hours after drug administration, all mice were humanely were killed and GFP+ MM bone lesions were visualized during necropsy by whole-body fluorescence imaging and harvested for further molecular analyses. (A) Flow cytometric analysis documents significant suppression of IGF-1R cell surface expression in MM tumor cells from 17-AAG-treated mice, compared to control mice. Filled curves correspond to staining with anti-IGF-1R antibody; open curves, staining with isotype control. (B) Hierarchical clustering analysis of in vivo gene expression profiles of MM-1S cells in 17-AAG-treated versus control mice.

Figure 6.

Figure 6.

Antiangiogenic effects of hsp90 inhibiton. (A) 17-AAG abrogates VEGF + bFGF-induced endothelial cell proliferation. (B) 17-AAG (1 μM, 3-day incubation) suppresses the survival of human microvascular endothelial cells (HMVECs), as evidenced by MTT colorimetric survival assay. (C) 17-AAG (1 μM for 24 hours) suppresses the surface expression of IGF-1R in HMVECs. Filled curves correspond to staining with anti-IGF-1R antibody; open curves, staining with isotype control. (D) 17-AAG (500 nM for 24 hours) abrogates constitutive and IGF-1-induced VEGF production by MM cells. (E) 17-AAG suppresses the transcriptional activity of HIF-1α in primary MM cells. Error bars indicate SD.

Figure 7.

Figure 7.

Hsp90 inhibition sensitizes tumor cells to other anticancer therapies. The in vitro anti-MM activities of doxorubicin (Doxo, 50 ng/mL, 48 hours, A) and bortezomib (PS-341, 5 nM, 24 hours, B) are enhanced by 17-AAG treatment (0.75 μM for the final 24 hours of each incubation) in primary MM tumor cells that are resistant to cytotoxic chemotherapy and bortezomib. Error bars indicate SD.

Figure 8.

Figure 8.

Molecular basis of hsp90 inhibitor-induced sensitization of MM cells to proteasome inhibitor bortezomib. In vitro treatment of chemo- and bortezomib-resistant primary MM tumor cells with bortezomib (PS-341, 5 nM, 12 hours), 17-AAG (250 nM, 12 hours) or their combination indicates that the combination of the 2 drugs induces more pronounced (A) accumulation of ubiquitinated proteins, as shown by immunoblotting analysis, and (B) inhibition of proteasome activity, evidenced by 20S proteasome chymotryptic activity assay, than either drug alone. Error bars indicate SD.

Figure 9.

Figure 9.

Schematic representation of diverse intracellular signaling cascades which can be impacted upon hsp90 inhibition, and of examples of multiple molecular levels where these effects can occur (through effects on expression/function of hsp90 client proteins and/or their downstream effectors). Solid arrows indicate activation and dashed lines indicate inhibitory events. Hsp90 inhibitors of the ansamycin family specifically inhibit hsp90, but the highly pleiotropic roles of their target confer to this class of agents the advantage of simultaneous blockade, by a single chemical entity, of diverse pathways implicated in tumor cell proliferation, survival, drug resistance, and microenvironmental interactions. This multifactorial activity of hsp90 inhibitors provides a framework for use of these agents to counteract mechanisms of resistance to multiple other agents, and increase tumor cell sensitivity to diverse conventional and novel agents.

References

    1. Isaacs JS, Xu W, Neckers L. Heat shock protein 90 as a molecular target for cancer therapeutics. Cancer Cell. 2003;3: 213-217. - PubMed
    1. Xu W, Mimnaugh E, Rosser MF, et al. Sensitivity of mature Erbb2 to geldanamycin is conferred by its kinase domain and is mediated by the chaperone protein Hsp90. J Biol Chem. 2001;276: 3702-3708. - PubMed
    1. Xu W, Mimnaugh EG, Kim JS, Trepel JB, Neckers LM. Hsp90, not Grp94, regulates the intracellular trafficking and stability of nascent ErbB2. Cell Stress Chaperones. 2002;7: 91-96. - PMC - PubMed
    1. Ohara-Nemoto Y, Nemoto T, Sato N, Ota M. Characterization of the nontransformed and transformed androgen receptor and heat shock protein 90 with high-performance hydrophobic-interaction chromatography. J Steroid Biochem. 1988;31: 295-304. - PubMed
    1. Solit DB, Zheng FF, Drobnjak M, et al. 17-Allylamino-17-demethoxygeldanamycin induces the degradation of androgen receptor and HER-2/neu and inhibits the growth of prostate cancer xenografts. Clin Cancer Res. 2002;8: 986-993. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources