Molecular chaperone gp96 is a novel therapeutic target of multiple myeloma - PubMed (original) (raw)

Molecular chaperone gp96 is a novel therapeutic target of multiple myeloma

Yunpeng Hua et al. Clin Cancer Res. 2013.

Abstract

Purpose: gp96 (grp94) is a key downstream chaperone in the endoplasmic reticulum (ER) to mediate unfolded protein response (UPR) and the pathogenesis of multiple myeloma is closely linked to dysregulated UPR. In this study, we aimed to determine the roles of gp96 in the initiation and progression of multiple myeloma in vivo and in vitro.

Experimental design: We generated a mouse model with overexpression of XBP1s and conditional deletion of gp96 in B-cell compartment simultaneously to identify the roles of gp96 in the development of multiple myeloma in vivo. Using a short hairpin RNA (shRNA) system, we silenced gp96 in multiple human multiple myeloma cells and examined the effect of gp96 knockdown on multiple myeloma cells by cell proliferation, cell-cycle analysis, apoptosis assay, immunohistochemistry, and human myeloma xenograft model. The anticancer activity of gp96 selective inhibitor, WS13, was evaluated by apoptosis assay and MTT assay.

Results: Genetic deletion of gp96 in XBP1s-Tg mice attenuates multiple myeloma. Silencing of gp96 causes severe compromise in human multiple myeloma cell growth through inhibiting Wnt-LRP-survivin pathway. We also confirmed that knockdown of gp96 decreased human multiple myeloma growth in a murine xenograft model. The targeted gp96 inhibitor induced apoptosis and blocked multiple myeloma cell growth, but did not induce apoptosis in pre-B leukemic cells. We have demonstrated that myeloma growth is dependent on gp96 both genetically and pharmacologically.

Conclusions: gp96 is essential for multiple myeloma cell proliferation and survival, suggesting that gp96 is a novel therapeutic target for multiple myeloma. Clin Cancer Res; 19(22); 6242-51. ©2013 AACR.

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Conflict of interest statement

Disclosure of Conflicts of Interest: The authors declare no competing financial interests.

Figures

Figure 1. Gp96 is required for the maintenance but not the differentiation of plasma cells in XBP1s-Tg mice

(A) Representative FACS plots of B220−CD138+ plasma cells in the bone marrow of four types of age/gender-matched mice (WT-wild type mice; KO-CD19cregp96flox/flox mice; Tg-B cell specific XBP1s-Tg mice; TKO-B cell specific gp96 KO and XBP1s-Tg mice). Number represents % of boxed cells over total number of cells analyzed. (B) Quantification of plasma cells in both spleen and bone marrow of four types of mice. The reduction of plasma cells in TKO mice is statistically significant (n=5 per group, *p < 0.05, T-test). Error bars indicate standard error of mean. (C) In vitro differentiation of plasma cells from TKO B cells is not impaired. Splenic B cells were purified with CD19 microbeads, labeled with CFSE, followed by stimulation with either IL4 alone (top row) or IL4 in combination with anti-μ antibody, agonistic CD40 antibody and IL21 (bottom row). Three days later, plasma cells were defined by CD138 expression and CFSE dilution. Number represents percentage of cells in each quandrant.

Figure 2. Development of myeloma in XBP1-Tg mice was blocked in the absence of gp96

(A, B) Sera from 5–16 weeks (n=9) (A) and 20–29 weeks (n=10) (B) Tg and TKO mice were collected. Total levels of IgG1 and IgM were measured by ELISA. (C) Serum protein electrophoresis was performed. Shown is a piece of representative data demonstrating the loss of γ globulin fraction in TKO mice. (D) Quantification of γ globulin fraction over the total protein in the serum. (E) Frozen kidney sections from 37-week-old Tg and TKO mice were analyzed for glomerular immunoglobulin deposition by immunofluorescence using anti-mouse IgG, IgM, IgA, κ and λ Abs. Error bars indicate standard error of mean. *p<0.05; **p<0.01; ***p<0.001 (T-test).

Figure 3. Gp96 is essential for multiple myeloma cell proliferation and survival

(A) Comparison of gp96 expression level in MM patients by cDNA microarray analysis. (B) Immunoblot analysis of gp96 and β-actin in WT and gp96 KO pre-B leukemic cells as well as empty vector (EV) control and gp96 knockdown (KD) U266B1, RPMI8226, OPM1, and INA-6 MM cells. (C) The growth of EV and gp96 KD MM cells was assessed over 72h- 96h; WT and gp96 KO pre-B leukemic cells were assessed over 72 h. (D) Cell cycle analysis of EV and gp96 KD RPMI8226 cells by propidium iodide staining and flow cytometry. The fraction of cells in subG1, G1, S, G2 and over G2 was quantified by FlowJo and data was plotted. Error bars indicate standard deviation. *p<0.05; **p<0.01 (T-test). (E) Representative plots of EV control and gp96 KD RPMI8226 cells were analyzed by flow cytometry for necrotic cells (PI+). (F) SCID mice were challenged subcutaneously with 5×106 WT and gp96 KD RPMI8226 cells. The kinetics of tumor growth was measured using a digital caliper. (G) Weight of excised tumor at the end of the experiment on week 8 (left panel) and the rate of tumor formation (right panel). Error bars indicate standard error of mean. ***p<0.001 (ANOVA). (H) Xenograft tumors were stained for gp96 and class I molecule of human leukocyte antigen (HLA) by immunofluorescence. (Scale bar: 25 μm). (I) The apoptotic cells were detected by TUNEL staining in xenografted WT and gp96 KD MM. (Scale bar: 25 μm).

Figure 4. gp96 knockdown in MM cells decreases canonical Wnt signaling, reduced expression of survivin, and causes mitotic catastrophe

(A) Immunoblot analysis of gp96, LRP6, β-catenin, cyclin D1, C-myc, grp78 and β-actin from EV control and gp96 KD RPMI 8226 cell lysates. (B) EV control and gp96 KD RPMI 8226 cells were treated with TWS119 and Lithium Chloride (LiCl) and the cell growth was assessed at 72 hours. Cell number without inhibitors was set as 1. Error bars indicate standard deviation. *p<0.05 (T-test). (C) Immunoblot analysis of gp96, survivin, caspase 9 and β-actin from EV control and gp96 KD RPMI 8226 cell lysates. (D) Xenograft MM as in Figure 3 was stained for survivin by immunohistochemistry. (E) Representative images of Hema 3 staining on EV, gp96 KD, gp96 KD and survivin overexpression, and survivin KD RPMI 8226 cells. The arrows show multi-nucleation in gp96 KD and survivin KD cells. (F, G, H) Quantification of multi-nucleated cells from EV and gp96 KD RPMI 8226 cells (F), gp96 KD and overexpression of survivin on gp96 KD RPMI 8226 cells (G), and EV and survivin KD RPMI 8226 cells (H). The increase of multi-nucleated cells in gp96 KD and survivin KD cells (F, H) and the reduction of multi-nucleated cells in gp96 KD cells by overexpression of survivin (G) are statistically significant. Error bars indicate standard deviation. *p < 0.05, **p<0.01, ***p <0.001 (T-test).

Figure 5. Selective gp96 inhibitor WS13 inhibits growth of human MM cells and induces apoptosis

(A) Pre-B leukemic cells, RMPI 8226, MM.1S, MM.1R, JK-6L, INA-6, OPM1 and U266B1 MM cells, were treated with 5 μM WS13 or vehicle control for 24 hours followed by flow cytometry analysis for necrotic cells (PI+ Annexin V+) or apoptotic cells (PI- Annexin V+). (B) Pre-B leukemic cells RPMI and multiple human MM cells were treated with 5 μM WS13 or vehicle control for 24, 72 and 120 hours followed by quantification of live cells by MTT assay. Error bars indicate standard deviation. *p<0.05; **p<0.01; ***p<0.001 (T-test).

Figure 6. A model for the roles of gp96 in myeloma

gp96 is a critical chaperone for LRP6 and is therefore required for canonical Wnt signaling, leading to release of β-catenin from its destruction complex (dotted circle), accumulation of nuclear β-catenin and upregulation of Wnt targets such as survivin Deletion of gp96 compromises survivin expression, leading to its failure to safeguard mitotic spindles, and therefore initiation of apoptosis. The mechanism of XBP1s or other UPR sensors to induce gp96 expression in MM cells is not depicted.

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Molecular chaperone gp96 is a novel therapeutic target of multiple myeloma - PubMed (original) (raw)