Hypoxia-inducible hexokinase-2 enhances anti-apoptotic function via activating autophagy in multiple myeloma

Abstract

Multiple myeloma (MM) is an incurable hematopoietic neoplasm derived from plasma cells, and existing in the bone marrow. Recent developments in the field of myeloma onco-biology have enabled the use of proteasome inhibitors (PIs) as key drugs for MM. PIs can increase cell sensitivity to endoplasmic reticulum stress, leading to apoptosis of myeloma cells. PI cannot kill all myeloma cells, however; one reason of this might be activation of autophagy via hypoxic stress in the bone marrow microenvironment. Hypoxia-inducible gene(s) that regulate autophagy may be novel therapeutic target(s) for PI-resistant myeloma cells. Here, a hypoxia-inducible glycolytic enzyme hexokinase-2 (HK2) was demonstrated to contribute by autophagy activation to the acquisition of an anti-apoptotic phenotype in myeloma cells. We found that hypoxic stress led to autophagy activation accompanied by HK2 upregulation in myeloma cells. Under hypoxic conditions, HK2 knockdown inhibited glycolysis and impaired autophagy, inducing apoptosis. The cooperative effects of a PI (bortezomib) against immunodeficient mice inoculated with HK2-knocked down myeloma cells were examined and significant tumor reduction was observed. An HK2 inhibitor, 3-bromopyruvate (3-BrPA), also induced apoptosis under hypoxic rather than normoxic conditions. Further examination of the cooperative effects between 3-BrPA and bortezomib on myeloma cells revealed a significant increase in apoptotic myeloma cells. These results strongly suggested that HK2 regulates the activation of autophagy in hypoxic myeloma cells. Cooperative treatment using PI against a dominant fraction, and HK2 inhibitor against a minor fraction, adapted to the bone marrow microenvironment, may lead to deeper remission for refractory MM.

Keywords: HK2; autophagy; hypoxia; microenvironment; multiple myeloma.

FIGURE 1

Hypoxia‐inducible autophagy in MM. A, Photograph of electron microscopy for 2 primary MM samples and a myeloma cell line KMS‐11 cultured under normoxia or hypoxia (1% O₂) for 48 h. B, Flow cytometry assay of autophagosome stained with DAPGreen in patient samples (n = 2) and the indicated 4 myeloma cell lines cultured under normoxia or hypoxia (1% O₂) for 48 h. y‐axis: cell count, x‐axis: autophagosome‐FITC

FIGURE 2

HK2 is upregulated through HIF activation in hypoxia‐stressed myeloma cells. A, Volcano plot showing the distribution of differentially expressed genes between primary MM samples (n = 4) cultured in hypoxia (1% O₂) vs normoxia for 48 h (GSE80545). Red dots illustrate transcripts significantly upregulated by hypoxia (fold change >2.0, P < .05). B, Gene Ontology (GO) analysis of hypoxia‐induced genes (red dots in A). C, Heat map of 15 probes (13 genes) including GO “glycolytic process” of (B). D, Scheme of the glycolytic process. Red arrows: genes upregulated by hypoxia displayed in (C). E, Gene expression change of HK1, HK2, HK3, and HK4 in patient samples cultured in hypoxia (1% O₂) vs normoxia for 48 h (GSE80545). F, qRT‐PCR of HK2 for cell lines (U266, KMS‐11, and MM.1S) cultured under hypoxia (1% O₂), serum starvation, with recombinant IL‐6 (4 ng/mL), co‐cultured with a stromal cell line HS‐5 (only U266) for 24 h. G, qRT‐PCR of HK2 for cell lines (U266, KMS‐11, and MM.1S) transiently transduced with siHIF1A and/or siHIF2A and control scrambled siRNA and cultured in normoxia or hypoxia (1% O₂) for 48 h. H, Western blot analysis of HK2 for primary samples (n = 2) and cell lines (KMS‐11, MM.1S, U266, RPMI‐8226, KMS‐12‐PE, and H929) cultured in normoxia or hypoxia (1% O₂) for 48 h. H, hypoxia; N, normoxia. Asterisks indicate statistical significance: *.01 ≤ P < .05; **.001 ≤ P < .01; ***P < .001; NS, not significant

FIGURE 3

HK2 knockdown leads myeloma cells to induce apoptosis under chronic hypoxia. A, qRT‐PCR analysis of HK2 for KMS‐11 and MM.1S cell lines transiently transduced with siHK2 #1, siHK2 #2, and control scrambled siRNA (Scr). B, Western blot analysis of HK2 for KMS‐11 and MM.1S cell lines transiently transduced with siHK2 #1, siHK2 #2, and control scrambled siRNA (Scr). C, Lactate concentration by ELISA assay and pH measurement for KMS‐11 cell line transiently transduced with siHK2 #1, siHK2 #2, and control scrambled siRNA (Scr). Cells were cultured at 1 × 10⁶/3 mL in normoxia or hypoxia (1% O₂) for 48 h. D, Apoptosis assay of KMS‐11 and MM.1S cell lines transiently transduced with siHK2 #1, siHK2 #2, and control scrambled siRNA (Scr) and cultured in normoxia or hypoxia (1% O₂) for 96 h. Left panel: x‐axis: Annexin V; y‐axis: 7‐AAD. Right panel: apoptotic cell rates of indicated cell lines were shown. Asterisks indicate statistical significance: *.01 ≤ P < .05; **.001 ≤ P < .01; ***P < .001; NS, not significant

FIGURE 4

HK2 knockdown suppresses hypoxia‐induced autophagy. A, Western blot analysis of LC3‐I, LC3‐II, and p62 for KMS‐11 transiently transduced with siHK2 #1, siHK2 #2, and control scrambled siRNA (Scr) and cultured in normoxia or hypoxia (1% O₂) for 48 h (left panel). LC3‐II/LC3‐I ratio of the western blot analysis is also shown (right panel). B, ELISA assay of p62 for KMS‐11 transiently transduced with siHK2 #1, siHK2 #2, and control scrambled siRNA (Scr) and cultured in normoxia or hypoxia (1% O₂) for 48 h. Bars represent mean ± 95% CI of 3 replicates. C, Photograph of electron microscopy for KMS‐11 transiently transduced with siHK2 #1 or control scrambled siRNA (Scr) and cultured in normoxia or hypoxia (1% O₂) for 48 h (left panel). Count of autophagosome/cell and lysosome/cell of siHK2‐ or control‐KMS‐11 (n = 10 each) is also shown (right panel). D, Flow cytometry assay of autophagosome stained with DAPGreen for KMS‐11 cell line cultured under hypoxia (1% O₂) for 48 h with or without AZD8055 (100 nmol/L, 6 h). y‐axis: cell count, x‐axis: autophagosome‐FITC (left panel). Mean fluorescence intensity (MFI) of each samples are shown (right panel). E, Schematic illustrations of effects of hypoxic stress (black arrows), siHK2 (red arrows) and the mTOR inhibitor AZD8055 (blue arrows) on the HK2‐mTORC1‐autophagy axis. The left panel shows that hypoxia‐inducible HK2 activates autophagy via mTORC1 inhibition, and the right panels shows that siHK2 inhibits autophagy and that the mTORC1 inhibitor AZD8055 activates autophagy. Asterisks indicate statistical significance: *.01 ≤ P < .05; **.001 ≤ P < .01; ***P < .001; NS, not significant

FIGURE 5

Cooperative effect of HK2 knockdown and proteasome inhibitor in vivo. A, qRT‐PCR analysis of HK2 for KMS‐11 stably transduced with shHK2 #A, #B, #C, #D, and control scrambled shRNA (Scr). B, Western blot analysis of HK2 for KMS‐11 stably transduced with shHK2 #A, #B, #C, #D, and control scrambled shRNA (Scr). C, qRT‐PCR analysis of HK1, HK2, HK3, and HK4 for KMS‐11 stably transduced with shHK2 #D or control scrambled shRNA (Scr). D, Illustration of the protocol of the in vivo transplantation and treatment; 1 × 10⁶ of shHK2 #D or control shRNA stably transduced KMS‐11 were inoculated into NOG mice. Mice were treated with bortezomib (1.0 mg/kg) or phosphate‐buffered saline intraperitoneally. Scr‐vehicle; n = 8, Scr‐BTZ; n = 10, shHK2‐vehicle; n = 8, and shHK2‐BTZ; n = 10. BTZ, bortezomib. E, Tumor growth curves of each groups are shown. x‐axis, days after transplantation (days); y‐axis, tumor volume (mm³, major × minor²/2). Right panel: rates of tumor volume reduction by bortezomib (BTZ) administration for the Scr group and shHK2 group. F, Photograph of tumors from each group are shown. G, Tumor weights of each groups are shown. Asterisks indicate statistical significance: *.01 ≤ P < .05; ***P < .001; NS, not significant.

FIGURE 6

Complementary effect of bortezomib and 3‐BrPA under chronic hypoxia in vitro. A, Apoptosis assay of KMS‐11, MM.1S, and normal peripheral blood mononuclear cells (PBMC). Cells were cultured in normoxia or hypoxia (1% O₂) for 48 h, and then 3‐bromopyruvate (3‐BrPA; 0, 20, 50, or 100 µmol/L) was added in the medium during 24 h. After the treatment, apoptosis assay was conducted. Left panel: x‐axis: Annexin V; y‐axis: 7‐AAD. B, Apoptosis assay of indicated cell lines and a refractory MM sample. Cells were cultured in normoxia or hypoxia (1% O₂) for 24 h, and then bortezomib (for cell lines: 10 nmol/L, for patient sample: 50 nmol/L) and/or 3‐BrPA (20 µmol/L) were added in the medium during 24 h. After the treatment, apoptosis assay was conducted. Upper left panel: x‐axis: Annexin V; y‐axis: 7‐AAD. Asterisks indicate statistical significance: *.01 ≤ P < .05; **.001 ≤ P < .01; ***P < .001; NS, not significant

FIGURE 7

Schematic illustration of the role of autophagy and HK2 in myeloma pathogenesis and hypoxia‐targeting therapy. A, Contribution of HK2‐autophagy pathway for cell survival of myeloma in hypoxic microenvironment. B, Complementary effect of conventional therapy and hypoxia‐targeting therapy