Hexokinase 2 displacement from mitochondria‐associated membranes prompts Ca ²⁺ ‐dependent death of cancer cells

Abstract

Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca 2+ overload caused by Ca 2+ release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca 2+ entry through plasma membrane. This results in Ca 2+ -dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.

ORAI2 Down-Regulation Potentiates SOCE and Decreases Aβ42 Accumulation in Human Neuroglioma Cells

“…While no difference was found in cells bathed in mKRB containing CaCl 2 (1 mM) (CTRL = 100 ± 7%, n = 26; ORAI2 = 112 ± 6%, n = 31, p = 0.11), ORAI2 overexpression almost doubled the resting Ca 2+ level of cells bathed in KCl-mKRB, containing high CaCl 2 (10 mM) (CTRL = 100 ± 13%, n = 31; ORAI2 = 176 ± 14.0%, n = 32, p < 0.001). However, due to the intrinsic low sensitivity of cytAEQ in reporting resting Ca 2+ levels, this issue was also investigated with nuclear-targeted GCaMP6 (H2B-GCaMP6), a probe with higher Ca 2+ affinity with respect to cytAEQ and a very large dynamic range (K d = 375 nM) [ 65 ], whose signal is synchronized with the cytosolic one [ 66 ]. In mKRB containing CaCl 2 (1 mM), ORAI2 overexpression caused a large increase in basal Ca 2+ levels ( Figure S6A ), as estimated by the ratio change occurring at rest upon addition of EGTA ( ΔR = R Ca −R EGTA ), being equal to 0.7 ± 0.2 and 12.4 ± 1.7 for CTRL and ORAI2 expressing cells, respectively ( p < 0.001).…”

Chemical Modulation of Mitochondria–Endoplasmic Reticulum Contact Sites

“…Such molecules include antimycotic drug clotrimazole [83], pyruvate analogue 3-bromopyruvate [84], and the plant stress hormone methyl jasmonate, which all induce detachment of HK from VDAC and stimulate apoptosis [85]. In addition, a selective peptide was shown to dislocate HK from MAMs in colon and breast cancer cells, and consequently to induce mitochondria Ca 2+ overload [86]. With respect to ANT, its association with VDAC is disrupted by arsenites, ionidamine, and steroid analogs [87].…”

The Two-Way Relationship Between Calcium and Metabolism in Cancer

“…The relationship between glycolysis and Ca 2+ is complex. In addition to the ATP-dependent regulation of Ca 2+ pumps, the physical association of glycolytic intermediates with Ca 2+ transporters modulates Ca 2+ homeostasis, while changes in the subcellular localization of glycolytic enzymes influence cell fate by impacting Ca 2+ homeostasis (Ciscato et al, 2020). Future investigations should focus on the cellular compartmentalization of metabolic intermediates as demonstrated for Ca 2+ .…”

“…In several cancer cells, the specific localization of the glycolytic enzyme hexokinase-2 (HK2) at mitochondria-ER contact sites [called mitochondria-associated ER membranes (MAMs)] prevents mitochondrial Ca 2+ overload, permeability transition pore (mPTP) opening and cell death by preventing ER Ca 2+ release through IP3R (Ciscato et al, 2020; Figure 1A). HK2 can also bind VDAC in the outer mitochondrial membrane (OMM) (Azoulay-Zohar et al, 2004;Shoshan-Barmatz et al, 2009).…”