Tumor Cells Switch to Mitochondrial Oxidative Phosphorylation under Radiation via mTOR-Mediated Hexokinase II Inhibition - A Warburg-Reversing Effect

<div><p>A unique feature of cancer cells is to convert glucose into lactate to produce cellular energy, even under the presence of oxygen. Called aerobic glycolysis [The Warburg Effect] it has been extensively studied and the concept of aerobic glycolysis in tumor cells is generally accepted. However, it is not clear if aerobic glycolysis in tumor cells is fixed, or can be reversed, especially under therapeutic stress conditions. Here, we report that mTOR, a critical regulator in cell proliferation, can be relocated to mitochondria, and as a result, enhances oxidative phosphorylation and reduces glycolysis. Three tumor cell lines (breast cancer MCF-7, colon cancer HCT116 and glioblastoma U87) showed a quick relocation of mTOR to mitochondria after irradiation with a single dose 5 Gy, which was companied with decreased lactate production, increased mitochondrial ATP generation and oxygen consumption. Inhibition of mTOR by rapamycin blocked radiation-induced mTOR mitochondrial relocation and the shift of glycolysis to mitochondrial respiration, and reduced the clonogenic survival. In irradiated cells, mTOR formed a complex with Hexokinase II [HK II], a key mitochondrial protein in regulation of glycolysis, causing reduced HK II enzymatic activity. These results support a novel mechanism by which tumor cells can quickly adapt to genotoxic conditions via mTOR-mediated reprogramming of bioenergetics from predominantly aerobic glycolysis to mitochondrial oxidative phosphorylation. Such a “waking-up” pathway for mitochondrial bioenergetics demonstrates a flexible feature in the energy metabolism of cancer cells, and may be required for additional cellular energy consumption for damage repair and survival. Thus, the reversible cellular energy metabolisms should be considered in blocking tumor metabolism and may be targeted to sensitize them in anti-cancer therapy.</p></div

Aerobic glycolysis [Warburg effect] was detected in human, mouse and xenograft tumors without mitochondrial mTOR localization.

<p>(A) Cellular mitochondrial ATP production and (B) lactate production and (C) mTOR western blotting in total cell lysates and mitochondrial fractions in HCT116 cells, 4T1 cells and SKOV3 xenograft.</p

Mitochondrial adaptive bioenergetics response was blocked by inhibiting the interaction of mTOR/Hexokinase II [HK II].

<p>(A) Co-immunoprecipitation of mTOR and HK II of MCF-7 cells treated with sham, IR with or without rapamycin. Cells were collected 24 h post-IR and IgG replaced anti-mTOR as a control. (B) HK II activity was measured in MCF-7 cells with the same treatment. (C) HK activity and mitochondrial ATP were measured in MCF-7 cells treated with sham, 24 h post-IR with DMSO and 24 h post-IR with the HK II inhibitor 3-BrPA. (D) Co-immunoprecipitation of HK II and MnSOD in MCF-7 cells treated with sham, 24 h post-IR, and 24 h post-IR with rapamycin pre-treatment. MnSOD activity was measured in MCF-7 cells at 24 h post-IR and 24 h post-IR with rapamycin. Data are mean ± SEM, p-values were included in each data.</p

Radiation enhanced mitochondrial mTOR accumulation and switched bioenergetics from glycolysis to mitochondrial respiration.

<p>(A) Human breast cancer MCF-7 cells were treated with sham or radiation (5 Gy IR) and samples collected at the indicated time points to measure (A) oxygen consumption and (B) mitochondrial ATP production. (C) Western blotting of total cell lysate or mitochondrial fraction was performed to detect mTOR localization following sham or radiation. (D) Flow cytometry was performed to determine the percentage of G2/M arrest. (E) Lactate production in MCF-7 cells was measured by lactate assay kit. Data are mean ± SEM, p values were included in each data.</p

Radiation enhanced mitochondrial mTOR and mitochondrial respiration in other human cancer cells.

<p>(A) Human colon cancer HCT116 and (B) brain tumor U87 cells were irradiated or sham irradiated with IR and collected at indicated time points. Mitochondrial ATP production and oxygen consumption were measured to determine the enhancement of mitochondrial functions after radiation. (C) Western blotting of mitochondrial fractions of HCT116 and U87 were performed to detect mTOR localization at time points shown after radiation. Data are mean ± SEM, p-values were included in each data.</p

Mitochondrial mTOR contributed to mitochondrial respiration.

<p>mTOR western blotting was performed using total cell lysates or mitochondrial fractions in paired normal and breast cancer MCF-10A, MCF-7 cells (A) and paired normal and transformed prostate cancer 267B1, 267B1/Ki cells (B). Cellular mitochondrial ATP production, oxygen consumption and lactate production of (C) MCF-10A and MCF-7, (D) 267B1 and 267B1/Ki cell lines were measured to compare the bioenergetics difference in two pairs of cells. Data are mean ± SEM, p values were included in each data.</p

A proposed mechanism switching from glycolysis to mitochondrial oxidative phosphorylation by mTOR-mediated Hexokinase II [HK II] inhibition in tumor cells under genotoxic stress condition created by ionizing radiation (IR).

<p>In tumor cells, radiation induces the localization of mTOR from cytosol to mitochondrial surface where it interacts and inhibits the activity of Hexokinase II. The mitochondrial accumulated mTOR, as a result, will reduce aerobic glycolysis. Meanwhile, the mTOR-mediated HK II inhibition will reduce the HK II-mediated mitochondrial suppression, enhancing mitochondrial oxidative respiration. Such switch of aerobic glycolysis to oxidative phosphorylation in cancer cells under acute genotoxic stress conditions represents a stress-adaptive feature of tumor cells and may links to tumor resistance to anti-cancer therapy.</p

Rapamycin inhibited mitochondrial mTOR and blocked radiation-induced bioenergetics switching.

<p>(A) Western blot of mTOR with mitochondria isolated from MCF-7 cells treated with 20 ng/mL rapamycin for 30 min followed by IR. (B) 3D Images of co-localization of TOM40 (green), a mitochondrial outer membrane protein, and mTOR (red) in MCF-7 cells 24 h after treatment with sham, IR or radiation plus rapamycin (IR+Rap). (C) Mitochondrial oxygen consumption, (D) ATP generation, (E) lactate production and (F) clonogenic survival assay at 24 h post-IR in DMSO and rapamycin treated MCF-7 cells. Data are mean ± SEM, p-values were included in each data.</p

A Facile and Versatile Post-Treatment Method to Efficiently Functionalize 3D-Printed Carbon Aerogels via Introducing Tailored Metal Elements

The rigorous viscoelastic rheology limits the ink formulation for 3D-printed carbon aerogels (CAs), making their functional customization challenging. Herein, we functionalize 3D-printed CAs via a unique combination of liquid phase deposition and thermal treatment. The electrodes’ improved areal capacitance after functionalization is 3949 mF cm–2, exceeding that of the pure 3D-printed CA electrode by 33.4%, which is superior by comparison with the carbonaceous electrodes reported previously. These impressive results highlight the effectiveness of this strategy for further functionalization of 3D-printed CAs. Moreover, numerous other potential applications, including catalysis, electromagnetic shielding, and solar steam generation, may be inspired by these findings

Effect of LJK-11 on tyrosine phosphorylation of signaling proteins.

<p>A549 cells were treated with 50 µM LJK-11 for 6, 12, 24, or 36 hours. The cell lysates were resolved by SDS-PAGE and analyzed by Western bolt analysis using antibodies as indicated. Antibodies specific to the phosphorylated forms of the indicated proteins are labeled with (-P).</p