Tohoku University Global COE Network Medicine Winter Camp of GCOE 2010 Feb, 5 th, 2011, Akiu, Sendai, Japan Novel functions of stanniocalcin-1(STC1) through Uncoupling Protein 2 (UCP2) up-regulation; Promoting Survival of Cancer Cells under Oxidative Stress and Inducing the Uncoupling Respiration (Warburg Effect). Respiratory Medicine, Graduate School of Medicine, Tohoku University Shinya Ohkouchi, Masahiko Kanehira, Toshiaki Kikuchi, Masahito Ebina and Toshihiro Nukiwa. We don’t have any relations with commercial companies in this study. Thank lots for their assistance, Dr Gregory J Block, Dr Darwin J Prockop, Center for Gene Therapy, Tulane University in New Orleans

What is uncoupling proteins (UCPs) and UCP2? There are five uncoupling proteins known in mammals (UCP1-5). UCP1 is expressed in brown fatty cell. UCP2 is expressed in ubiquitous tissue and cancer. Through upregulation of UCPs, the parts of protons gained from respiratory chain are used for other purposes than ATP synthesis. This phenomenon is called ‘proton leak’. Proton leak results in mitochondrial membrane potential (MMP, ΔѰM) reduction. Proton leak and ΔѰM reduction result in heat production (UCP1), ROS reduction, inducing uncoupling respiration (aerobic glycolysis, Warburg effect) and Promoting FFA (free fatty acid) utilization etc. Heat↑, ROS↓, Warburg effect, FFA utilization↑ Mitochondria membrane MMP=mitochondrial membrane potential H + =Proton Proton leak and ΔѰM↓ e-e- UCPs ⇑ Anaerobic Glycolysis without mitochondria ⇑ (ATP production ⇓,Lactate production ⇑ ) These processes need oxygen. Therefore, this is ‘Aerobic Glycolysis’. Make sense two Glycolysis, ‘Aerobic’ and ‘Anaerobic’. ATP production ⇑ Oxidative Phosphorylation

UCP2 and Aerobic Glycolysis (Uncoupling Respiration, Warburg Effect). Aerobic(mitochondria) Anaerobic (no mitochondria) Oxidative phosphorylation ⇓ Uncoupling Respiration ΔѰM↓ ROS↓ FFA Glutamine utilization↑ This status needs oxygen. Warburg Effect Definition; Glycolysis is also observed under normoxia. There are many controversy about interpretation of Warburg Effect. This figure shows my opinion. Oxygen consumption ⇒ or ⇑ Oxygen consumption ⇓ Glycolysis Some Cancers may prefer this environment shown in circle. Cancers get ‘more energy’. UCP2 induces Uncoupling Respiration. Aerobic Glycolysis are promoted by Uncoupling Respiration. This metabolic status composes a part of Warburg effect. (Cancer Research 2009 (69): 2163) Aerobic Glycolysis ≑ Uncoupling Respiration ≤Warburg Effect

Seahorse XF24 can differentiate two glycolysis status, uncoupling (aerobic) & anaerobic glycolysis. Uncoupler, ΔѰM ↓ Inhibit electron transport Seahorse XF24 bio-analyzer This machine can measure Oxygen Consumption Rate (OCR) and Extra-Cellular Acidification Rate (ECAR) of samples at the same time. Inhibit anaerobic metabolism Medium This is A549 data using XF24 by ourselves. Reagents induce specific metabolic status by each drugs. XF24 can measure and differentiate two glycolysis status. (ECAR; Lactate production) Clark oxygen electrode in dept of tech Clark oxygen electrode are usually used for measuring oxygen consumption however lactate production can not be measured at the same time. Our medical school doesn’t have both Clark oxygen electrode & XF24 bio-analyzer! OCR ⇓ ECAR ⇑ OCR ⇑ ECAR ⇑ OCR ⇑ ECAR ⇓ OCR; oxygen consumption)

What is STC-1 (Stanniocalcin-1)? Highly conserved homodimeric secreted glycoprotein. STC-1 was found out in fish gill firstly. (Mol Cell Endocrinology (1):7) Inhibitor of apoptosis of neuron & cardiomyocyte under ischemia. Inhibitor of apoptosis of neuron & cardiomyocyte under ischemia. (Proc Natl Acad Sci U S A (7):3637, Stem Cell (27): 670) STC1 transgenic mice are gluttonous but show dwarfed phenotype (energy wasting phenotype). (Endocrinology (3):868) STC1 transgenic mice are gluttonous but show dwarfed phenotype (energy wasting phenotype). (Endocrinology (3):868) Regulation of mitochondrial oxidative phosphorylation and Ca 2 + & PO 4 - transport. STC1 is a mitochondrial related hormone. (Mol Cell Endocrinol (1-2):90) Regulation of mitochondrial oxidative phosphorylation and Ca 2 + & PO 4 - transport. STC1 is a mitochondrial related hormone. (Mol Cell Endocrinol (1-2):90) We have shown STC1 secreted from MSCs ameliorate injured A549 under hypoxic and acidic condition. Oxidative stress is main source of cell stress in these situations. (Stem Cell (27): 670) We have shown STC1 secreted from MSCs ameliorate injured A549 under hypoxic and acidic condition. Oxidative stress is main source of cell stress in these situations. (Stem Cell (27): 670) Recent study showed STC1 activate mitochondrial anti-oxidant pathway via UCP2 upregulation. Therefore we hypothesized STC1 decrease intracellular ROS via UCP2 upregulation. (J Leukoc Biol 2009 (86): 981) Recent study showed STC1 activate mitochondrial anti-oxidant pathway via UCP2 upregulation. Therefore we hypothesized STC1 decrease intracellular ROS via UCP2 upregulation. (J Leukoc Biol 2009 (86): 981) We also evaluated which STC1 induce uncoupling respiration (Aerobic Glycolysis, a part of Warburg effect) in A549 cells via UCP2 upregulation.

Our Hypothesis STC1 secreted from MSCs ⇓ UCP2 up-regulation ⇓ ΔѰM reduction in A549 insulted by oxidant stress (H2O2 in our system) ⇓ ROS reduction and Uncoupling Respiration (Aerobic Glycolysis) ⇓ A549 survival Secretion of STC1 Unknown Secreted Factor from A549 Survival of A549 Injury (Hypoxia, Acidosis, H 2 O 2 →Oxidative Stress↑) MSCs Up- regulation of STC1 ROS ↓ Uncoupling Respiration (Warburg Effect) (aerobic glycolysis) A549 Up- regulation of UCP2 Reduction of ΔѰM

Method （ Cell impermeable ） Humoral factor can pass through the membrane. （ Stem Cell (27) p670.) 0.4um Transwell Membrane Oxidative Stress Inducer

Result 1. MSCs Reduced ROS-mediated Cell Death in A549s in an STC1 Dependent Manner. A Annexin/PI assay （ Flow Cytometry) Addition of anti-STC1 antibody abolished effects of MSCs to inhibit cell death of A549 in H 2 O 2. B Knocking down of STC1 with specific siRNA in MSCs abolished effects of MSCs to inhibit cell death of A549 in H 2 O 2. C D MSCs reduced cell death of A549 exposed to H 2 O 2. H 2 O 2 induce STC1 expression in MSCs. Recombinant STC1 inhibit cell death of A549 insulted by H 2 O 2.

Result 2. MSCs Reduced ROS Accumulation of A549 Exposed to H 2 O 2 in an STC1 Dependent Manner. Co-cultured MSCs on Transwell reduced ROS accumulation of A549 in H 2 O 2. ROS↑ A B C rSTC1 also reduced ROS accumulation of A549 in H 2 O 2. D Knocking down STC1 of MSCs with specific siRNA abolished the ability of MSCs to reduce ROS of A549 in H 2 O 2. rSTC1 also reduced ROS accumulation induced by paclitaxel. rSTC1 function as a scavenger is stronger than that of N-acetylcysteine (NAC).

Result 3. STC1 Increased UCP2 Expression in A549 Cells Exposed to H 2 O 2. Human rSTC1 increased UCP2 expression of A549 exposed to H 2 O 2 in both RT- PCR and Western blotting. Addition of anti-STC1 antibody decreased UCP2 expression in A549 co- cultured with MSCs. A B

When UCP2 was knocked down in A549 with specific siRNA, rSTC1 could not reduce ROS in A549 exposed to H 2 O 2. Quantification of A. A B Result 4. Reduction of ROS by STC1 was Dependent on UCP2 Upregulation.

Result5. STC1 Induced ΔѰM reduction and lactate production (glycolysis) in A549 cells. STC1 induced ΔѰM reduction and lactate production (glycolysis) under H 2 O 2 existence and normal culture condition in A549. Knocking down of UCP2 in A549 with specific siRNA resulted in up-regulation of ΔѰM. ΔѰM reduction of A549 is also observed in hypoxic condition. A B C MMP of A549 DMEM DMEM+rSTC1

Result 6. STC1 Induced uncoupling respiration (aerobic glycolysis, Warburg effect) in A549 cells. A B C Normal culture condition ≑ Aerobic glycolysis ≤ Warburg effect Time (min) Oxygen consumption rate (%) Extracellular acidification rate (%) (Lactate production rate) rSTC1 increases oxygen consumption (OCR) and lactate production (ECAR) in A549 under normal culture condition (Seahorse XF24 bio-analyzer). Oxygen consumption rate (%) (OCR) Extracellular acidification rate (%) (EACR) OCR and ECAR data of A are shown in same graph. This graph reveal STC1 induces uncoupling respiration in A549. Under oxidative stress, rSTC1 also increases oxygen consumption in A549. Oxygen consumptions were measured using Clark oxygen electrode. OCR ECAR OCR ECAR XF24 Clark electrode OCR ⇑ ECAR ⇑ OCR ⇑ ECAR ⇓ OCR ⇓ ECAR ⇑

Supplemental Result 1. ROS reduction and ΔѰM reduction induced by rSTC1 are also observed in other lung cancer cell lines. AB

Conclusion MSCs inhibit ROS accumulation and induce Uncoupling Respiration (Aerobic Respiration, Warburg Effect) in A549 through UCP2 upregulation by STC1 secretion. These data suggests MSCs give the advantage for survival to cancer cells under ROS stress and starvation in surrounding circumstance. STC1 may provide promising avenues for treatment of ROS and control of metabolic status in cancer, metabolic disorder etc. Secretion of STC1 Unknown Secreted Factor from A549 Survival of A549 Injury (Hypoxia, Acidosis, H 2 O 2 →Oxidative Stress↑) MSCs Up- regulation of STC1 ROS ↓ Uncoupling Respiration (Warburg Effect) (Aerobic Glycolysis) A549 Up- regulation of UCP2 Reduction of MMP PNAS in press.