Oxidative stress activates the TRPM2-Ca2 +-CaMKII-ROS signaling loop to induce cell death in cancer cells

    Oxidative stress activates the TRPM2-Ca 2 + -CaMKII-ROS signaling loop to induce cell death in cancer cells Qian Wang, Lihong Huang, Jianbo Yue PII: DOI: Reference:

S0167-4889(16)30339-1 doi:10.1016/j.bbamcr.2016.12.014 BBAMCR 18015

To appear in:

BBA - Molecular Cell Research

Received date: Revised date: Accepted date:

11 October 2016 5 December 2016 13 December 2016

Please cite this article as: Qian Wang, Lihong Huang, Jianbo Yue, Oxidative stress activates the TRPM2-Ca2 + -CaMKII-ROS signaling loop to induce cell death in cancer cells, BBA - Molecular Cell Research (2016), doi:10.1016/j.bbamcr.2016.12.014

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Oxidative stress activates the TRPM2-Ca2+-CaMKII-ROS signaling loop to induce cell death in

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cancer cells

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Department of Molecular Medicine and Pathology, University of Auckland, New Zealand

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Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China

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Qian Wang1, 2, Lihong Huang1, and Jianbo Yue1*

*Correspondence and requests for materials should be addressed to J Yue ([email protected])

Running title: TRPM2-Ca2+-CAMKII-ROS signaling loop in cell death

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ACCEPTED MANUSCRIPT ABSTRACT High intracellular levels of reactive oxygen species (ROS) cause oxidative stress that results in

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numerous pathologies, including cell death. Transient potential receptor melastatin-2 (TRPM2),

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a Ca2+-permeable cation channel, is mainly activated by intracellular adenosine diphosphate ribose (ADPR) in response to oxidative stress. Here we studied the role and mechanisms of

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TRPM2-mediated Ca2+ influx on oxidative stress-induced cell death in cancer cells. We found

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that oxidative stress activated the TRPM2-Ca2+-CaMKII cascade to inhibit early autophagy induction, which ultimately led to cell death in TRPM2 expressing cancer cells. On the other

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hand, TRPM2 knockdown switched cells from cell death to autophagy for survival in response to oxidative stress. Moreover, we found that oxidative stress activated the TRPM2-CaMKII cascade

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to further induce intracellular ROS production, which led to mitochondria fragmentation and loss of mitochondria membrane potential. In summary, our data demonstrated that oxidative stress

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activates the TRPM2-Ca2+-CaMKII-ROS signal loop to inhibit autophagy and induce cell death.

Keywords: TRPM2; Oxidative stress; Ca2+; Reactive oxygen species; CaMKII; autophagy

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ACCEPTED MANUSCRIPT INTRODUCTION Reactive oxygen species (ROS) mainly includes superoxide anion (O2-), hydroxyl radical (HO-),

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and non-radical hydrogen peroxide (H2O2). O2-, the main intracellular ROS, is generated by the

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mitochondrial electron transport chain during continuous aerobic respiration. O2- is only moderately reactive, but can be converted to H2O2 by superoxide dismutase and ultimately to

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highly toxic HO- by Fenton reaction. ROS can also be produced by membrane NADPH oxidase,

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peroxisomes, or the cytochrome P450 system in response to various stimuli, e.g. cytokines and cytotoxic drugs. High levels of ROS, caused by drugs or various pathological conditions, can

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damage lipids, proteins, and DNA to induce cell death. Cells, in turn, develop a complicated antioxidant defense mechanism to offset oxidative damage by strictly regulating ROS levels in

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vivo. A balance between ROS formation and antioxidant defense is essential to cell survival and growth, whereas an imbalance generates oxidative stress, which has been implicated in numerous

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human diseases [1, 2]. Notably, ROS can differentially regulate autophagy, and vice versa. Yet, the molecular mechanisms underlying the interplay between ROS and autophagy remain elusive [3].

Autophagy is an evolutionarily conserved catabolic degradation cellular process, whereby misfolded proteins or damaged organelles are sequestered by autophagosome, a doublemembrane vesicle. Autophagosome then fuses with lysosome to form an autolysosome, and the contents inside autophagosome are subsequently digested and recycled to maintain cellular homeostasis. A wide variety of stresses, e.g. nutrient starvation and oxidative stress, can markedly induce autophagy for cell survival. Autophagy is a double-edged sword for many cellular processes, depending upon the genetic background and microenvironment [4, 5].

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ACCEPTED MANUSCRIPT Dysregulated autophagy has been associated with a variety of human diseases, including cancers, neurodegenerative diseases, and infections [6-8].

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The initiation of autophagy is regulated by ULK1/ULK2 complexes and the class III

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phosphatidylinositol-3 kinase (PI3K) complexes. VPS34 binds with BECLN1, UVRAG, and VPS15 to produce phosphatidylinositol 3-phosphate (PtdIns3P), which subsequently recruits ATG

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process.

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other

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conjugation

of

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phosphatidylethanolamine (PE) to LC3 is also essential for the induction of autophagy, which is sequentially catalyzed by the protease ATG4, E1-like ATG7, and E2-like ATG3, which converts

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the cytosolic LC3 (LC3-I) to the autophagic vesicle-associated form (LC3-II) [6-8]. Transient potential receptor melastatin-2 (TRPM2), as a prominent oxidative stress sensor,

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is a non-selective Ca2+ permeable cation channel [9]. The main activator for TRPM2 is intracellular adenosine diphosphate ribose (ADPR), which binds to the C-terminus Nudix-like

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domain of TRPM2 to open it. Ca2+, cADPR, H2O2, and NAADP can positively modulate TRPM2, whereas AMP and acidic pH negatively regulate it [10]. TRPM2 channel has been indicated in a variety of cellular processes, e.g. insulin secretion, temperature homeostasis, etc. [11-13]. Oxidative stress could potently activate TRPM2 for Ca2+ influx, thus leading to cell death [14]. Paradoxically, oxidative stress can also activate autophagy to prevent the accumulation of ROS, thereby alleviating the damage for cell survival [3]. Here we examined the ability of oxidative stress to activate TRPM2-Ca2+ signaling to regulate cell death and autophagy in cancer cells with or without the expression of TRPM2. We found oxidative stress activated the TRPM2-Ca2+-CaMKII-ROS signaling loop, resulting in autophagy inhibition, mitochondria fragmentation, and ultimate cell death.

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ACCEPTED MANUSCRIPT RESULTS Requirement of TRPM2 in H2O2-meidated cell death induction in PC3 human prostate cancer

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cells. Human prostate cancer cells, e.g. PC3 cells, are sensitive to oxidative stress-induced cell death, and TRPM2 is expressed in these cells (Fig. S1A and S1B) [15-17]. We, thus, assessed

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the role of TRPM2-mediated Ca2+ influx in oxidative stress induced cell death in PC3 cells. As

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shown in Figure 1A, H2O2 addition triggered intracellular Ca2+ increase in a concentration

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dependent manner. TRPM2 knockdown markedly inhibited H2O2 (1 mM)-induced Ca2+ increase (Fig. 1B). As expected, H2O2 treatment induced cell death in a concentration dependently

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manner, whereas TRPM2 knockdown significantly mitigated H2O2-induced cell death (Fig. 1C). TUNEL assay further confirmed that H2O2 (0.5 mM) induced cell apoptosis in PC3 cells, which

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was significantly reduced by TRPM2 knockdown (Fig. 1D). Likewise, TRPM2 knockdown

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significantly suppressed the ability of H2O2 (25 M) to inhibit colony formation of PC3 cells

death.

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(Fig. S1C). Thus, these data confirm that TRPM2 is required for oxidative stress-induced cell

Role of autophagy in H2O2-induced cell death in cancer cells. Since ROS could induce autophagy for cell survival, we next assessed whether the autophagy activity is compromised upon H2O2 treatment in control PC3 cells. Indeed, H2O2 markedly decreased LC3-II levels in control PC3 cells but induced it in TPRM2 knockdown cells (Fig. 2A). H2O2-induced LC3-II decrease in PC3 cells was abolished by pretreatment with a TRPM2 antagonist, clotrimazole (CLT) (Fig. 2B). To further confirm the role of TRPM2 in H2O2-mediated autophagy inhibition in PC3 cells, we infected PC3 cells with lentiviruses carrying expression cassettes that encode tandem fluorescence-tagged LC3B (tfLC3B) [18]. The LC3-II positive autophagosomes are labeled with both GFP and RFP signals shown as yellow puncta, and after fusion with

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ACCEPTED MANUSCRIPT lysosomes, autolysosomes are shown as red only puncta because GFP loses its fluorescence in acidic pH. As shown in Figure S2A and S2B, rapamycin treatment greatly increased both yellow

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and red only puncta, yet treatment of cells with bafilomycin (BAF), an inhibitor of the vacuolar

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proton pump that blocks the fusion of autophagosomes with lysosomes [19], markedly induced the accumulation of yellow puncta only, indicating that the autophagy is arrested at

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autophagosomes. Thus, the tfLC3B-expressing PC3 cells can be used to monitor the progression

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of autophagy. Not surprisingly, H2O2 (0.5 mM) treatment did not affect either green or red LC3 puncta in control PC3 cells, whereas it markedly increased both the red and yellow LC3 puncta

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in TRPM2 knockdown cells (Fig. 2C). Moreover, in control BAF-treated PC3 cells, addition of H2O2 (0.5 mM) markedly decreased LC3-II puncta, whereas in BAF-treated TRPM2 knockdown

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PC3 cells, H2O2 further increased LC3-II puncta (Fig. S2C). Taken together, these results indicate that H2O2 induces cell death but inhibits autophagy in TRPM2 expressing PC3 cells; on

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the other hand, H2O2 induces autophagy for cell survival in TRPM2 knockdown PC3 cells. Obviously, these data hint that ROS can trigger Ca2+ influx via TRPM2 to affect cell fate under oxidative stress.

Role of TRPM2 in H2O2-mediated cell death and autophagy regulation in HeLa cells. In contrast to PC3 cells, HeLa cells do not express functional TRPM2, and it is not surprising that TRPM2 overexpression in HeLa cells [20] markedly increased the ability of H2O2-induced cell death (Fig. 3A-3C). This H2O2 (200 M)-induced cell death in HeLa cells was significantly inhibited by a TRPM2 inhibitor, CLT (Fig. 3B and 3C). As expected, H2O2 treatment markedly increased LC3II levels in control HeLa cells but decreased it in TRPM2-overexpressing HeLa cells in a concentration dependent manner (Fig. 4A), hinting that autophagy inhibition might be responsible for the increased vulnerability of cells to oxidative stress-induced cell death.

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ACCEPTED MANUSCRIPT Interestingly, we found that H2O2-mediated LC3-II changes were blocked by EGTA pretreatment (Fig. S3A), suggesting that Ca2+ is essential for ROS-mediated autophagy regulation. Along this

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line, numerous studies indeed have already documented that intracellular Ca2+ can differentially

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modulate autophagy upon the context of time, space, Ca2+ source, and cell status [21, 22]. To clarify the role of autophagy in oxidative stress-induced cell death, HeLa cells was

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treated with H2O2 in the presence or absence of BAF, which blocks the fusion of

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autophagosomes with lysosomes in HeLa cells (Fig. S3B). As shown in Fig. 4B and 4C, BAF treatment significantly increased H2O2 (200 M)-induced cell death in control, not TRPM2

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overexpressing, HeLa cells. To further assess the role of autophagy in in oxidative stress-induced cell death, ATG5 was knocked down in HeLa cells, which do not express functional TRPM2. As

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shown in Fig. 4D, ATG5 knockdown blocked H2O2 induced autophagy in HeLa cells. As expected, ATG5 knockdown significantly increased H2O2-induced cell death in control HeLa

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cells (Fig. 4E). Therefore, it is clear that autophagy inhibition promotes oxidative stress-induced cell death in cancer cells. These data also echo that activation of the ROS-TRPM2-Ca2+ cascade inhibits early autophagy induction, which is at least partially responsible for ROS-induced cell death.

H2O2 triggered mitochondria fragmentation in TRPM2-expressing cancer cells. Physiological level of ROS is involved in many cellular processes, whereas excess ROS is harmful [23]. We thus checked the effects of TRPM2-mediated Ca2+ influx on intracellular ROS levels. As shown in Figure 5A, ROS level in PC3 cells was similar to TRPM2 knockdown cells, but H2O2 treatment induced much less increase of intracellular ROS in TRPM2-knockdown cells than that in control cells. Likewise, the intracellular ROS level in TRPM2-overexpressing HeLa cells was similar to that in control cells, and H2O2 treatment increased ROS levels in both control and

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ACCEPTED MANUSCRIPT TRPM2 expressing cells, with the latter level being significantly higher (Fig. 5B). Pretreatment of HeLa cells with BAPTA-AM, a Ca2+ chelator, not only significantly inhibited H2O2-induced

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cytosolic ROS production, but also abolished the enhancive effects of TRPM2 on intracellular ROS increases (Fig. S4A). These data indicated that cytosolic Ca2+ is involved in ROS

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production in cells. Notably, the mutual interaction between Ca2+ and ROS has been well

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documented [24]. In addition, these data suggested that H2O2 activates TRPM2 to trigger Ca2+

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influx, which subsequently contribute to intracellular ROS increase, thereby constituting a positive feedback loop. On the other hand, treatment of cells with BAF, an autophagy inhibitor,

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failed to affect the enhancive effects of TRPM2 on ROS increase (Fig. S4A), suggesting that autophagy inhibition is not responsible for the TRPM2-mediated ROS increase.

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Since ROS normally causes cell death by activating the intrinsic cell death pathway through mitochondria, we subsequently assessed the effects of H2O2 on mitochondria in cancer

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cells with or without TRPM2 expression. As expected, H2O2 (200 M)-induced mitochondria fragmentation in TRPM2 knockdown PC3 cells was much less severe than that in control PC3 cells (Fig. 5C). Similarly, H2O2 (200 M) induced mitochondria fragmentation in TRPM2overexpressing, not control, HeLa cells (Fig. 5D and S4B).

Interestingly, mitophagy was

induced by H2O2 (75 M) treatment in control HeLa cells (Fig. S4C). Moreover, H2O2 (200 M) treatment quickly abolished mitochondrial membrane potential in control, not TRPM2 knockdown, PC3 cells (Fig. 5E), and in TRPM2-overexpressing, not control, HeLa cells (Fig. 5F). These results indicate that oxidative stress activates TRPM2 for Ca2+ influx to induce further ROS production in a positive feedback loop, which results in damaged mitochondria and ultimately leads to cell death.

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ACCEPTED MANUSCRIPT Requirement of CaMKII in oxidative stress-induced cell death in PC3 cancer cells. We have previously shown that oxidative stress activates CaMKII via TRPM2-mediated Ca2+ influx in

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TRPM2-expressing HeLa cells [20]. Interestingly, auto-phosphorylation of CaMKII was not

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affected by H2O2 treatment in PC3 cells as shown by the immunoblot analysis with an antiphospho-CAMK2-Thr286/287 antibody (Fig. 6A). Yet, H2O2 markedly induced the oxidation of

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CaMKII in control PC3 cells, and TPRM2 knockdown abolished it (Fig. 6B) as shown by the

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immunostaining analysis with an anti-oxidized CAMK2-(C) M281/282 antibody. As expected, pretreatment of PC3 cells with AIP, an CaMKII inhibitor, reversed H2O2-meidated LC3-II

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decrease in PC3 cells (Fig. 6C). Likewise, treatment of cells with AIP or expression of a dominant negative mutant of CaMKII, CaMK2AK42R, in PC3 cells significantly mitigated H2O2-

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induced cell death (Fig. 6D, 6E, and S5). Moreover, treatment of PC3 cells or TRPM2overexpressing HeLa cells with CaMKII inhibitors, AIP or KN93, abolished H2O2-induced

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intracellular ROS increases (Fig. 7A and 7B). As positive controls, pretreatment of cells with an TRPM2 antagonist, CLT, or antioxidant, N-acetylcysteine (NAC), also abolished H2O2-induced intracellular ROS increases in TRPM2-overexpressing HeLa cells (Fig. 7B). Not surprising, H2O2 treatment failed to abolish the mitochondrial membrane potentials in PC3 cells pretreated with a CaMKII inhibitor, AIP (Fig. 7C). Taken together, these data indicate that oxidative stress activates the TRPM2-Ca2+-CaMKII-ROS signal loop to induce cell death.

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ACCEPTED MANUSCRIPT DISCUSSION Here we found that oxidative stress activated TRPM2-Ca2+-CaMKII signaling cascade to further

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induce intracellular ROS production, constituting a positive feedback loop, thereby inhibiting

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autophagy and inducing cell death. On the other hand, in the absence of TRPM2-mediated Ca2+ influx, oxidative stress induced autophagy for cell survival. In summary, our data clearly

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establish the TRPM2-Ca2+-CaMKII-ROS signaling loop as the molecular switch determining cell

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to live or die in response to oxidative stress.

Tumor cells increase the production of intracellular ROS and/or consumption of

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antioxidant proteins, thereby resulting in sustained higher levels of ROS than normal cells [25]. The tumor cells originally are particularly sensitive to oxidative stress. In fact, many

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chemotherapy and radiotherapy markedly induce oxidative stress in tumors, and the resultant high levels of ROS add up cytotoxicity of these treatments on tumors. Yet, many of tumors

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gradually develop ways to cope with oxidative stress and ultimately grow resistance to the treatments. In these tumors, oxidative stress actually induces autophagy for survival instead of cell death [26]. Coincidentally, TRPM2 is not functioning in many cancer cells, either due to the lack of expression of full length or the expression of short dominant negative form of TRPM2 [27]. Multiple TRPM2 splice variants, e.g. TRPM2-S, which is a short splice variant of TRPM2, TRPM2-AS, a natural antisense transcript, and TRPM2-TE, a truncated transcript, have been identified in melanoma, breast cancer, lung tumor, and other tumors. These splice variants inhibit full-length TRPM2 activity, and restoring TRPM2 functions in these tumors re-sensitizes these tumor to oxidative stress-induced cell death [16, 28, 29]. Notably, fluorouracil (5-FU), an inhibitor of thymidylate synthase commonly used to treat anal, breast, pancreatic, and skin cancer [30, 31], exhibited low cytotoxicity in PC3 cells, but combination of 5-FU and lower

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ACCEPTED MANUSCRIPT concentration of H2O2 synergistically decreased cell viability in control, not TRPM2 knockdown, PC cells (Fig. S6A). Our current data also suggest that oxidative stress generated by the anti-

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cancer therapy might activate autophagy for cell survival in the absence of TRPM2-mediated

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Ca2+ influx (Fig. S6B and data not shown), thereby enabling the tumor cells to escape therapy. Therefore, restoring TRPM2 expression or application of a CaMKII agonist or autophagy

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inhibitor in these tumors should re-sensitize them to the treatment. Along this line, numerous

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preclinical studies have found that the inhibition of autophagy restores chemosensitivity and promotes tumor cell death by diverse anticancer therapies [32].

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Interestingly, ROS induces Ca2+ influx via TRPM2, which in turn contributes to additional intracellular ROS production, thus constituting a positive-feedback loop (Fig. 5A, 5B, 7A, 7B,

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and S4A). The sources of the additional ROS induced by TRPM2-mediated Ca2+ influx remain

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unknown. It is possible that the Ca2+-influx induced ROS is from the damaged mitochondria

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because of apoptosis induction. Yet, in the absence of TRPM2-mediated Ca2+ influx, autophagy inhibition switched cells to apoptosis upon ROS treatment (Fig. 4B, 4C, and 4E) but failed to produce additional intracellular ROS (Fig. S4A), arguing that the additional ROS induced by Ca2+ influx is the cause, not the result, of the autophagy inhibition and apoptosis induction. Other than mitochondria, NOX family of ROS-generating NADPH oxidases is another major ROS source [33], and NOX activity can be modulated membrane potential [34]. Thus, it is possible that TRPM2-mediated Ca2+ influx depolarizes plasma membrane, which subsequently activates NOX to increase intracellular ROS levels [35]. Recently, it has been shown that TRPM2 directly interacts with RAC1, a component of the NADPH oxidase complex, to increases oxidant stress in ischemic kidney injury [36]. Whether other NADPH oxidase complex members can be modulated by TRPM2/Ca2+ cascade remains to be determined.

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ACCEPTED MANUSCRIPT Importantly, we found that inhibiting CaMKII abolished H2O2-induced intracellular ROS increases (Fig. 7A and 7B), indicating that CaMKII is required for this positive-feedback loop.

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We are currently in the process of identifying the downstream targets of CaMKII for ROS

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production and cell death induction. Along this line, it has been shown that inhibition of PIM kinase prevent nuclear accumulation of Nrf2, leading to ROS increase and p38 and mTOR

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activation [37, 38]. Therefore, it is of interest to assess the cross-talk between CaMKII and Pim

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kinase.

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ACCEPTED MANUSCRIPT MATERIALS AND METHODS Cell culture- HeLa cells (ATCC) were maintained in Dulbecco's Modified Eagle Medium (DMEM,

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powder, Invitrogen) with 10% Fetal Bovine Serum (FBS, Invitrogen) and 1% penicllin-streptomcyin

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(P/S, Invitrogen). Human prostate cancer cell line PC3 cell line, provided by Professor Yuen-Chiu Chan of the University of Hong Kong, was maintained in RPMI-1640 Medium (Invitrogen) with 10%

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Fetal Bovine Serum (FBS, Invitrogen) and 1% penicllin-streptomcyin (Invitrogen).

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shRNA and lentivirus production and infection- The lentivirus production and infection were performed as described previously [20]. Briefly, two 21-mers in human TRPM2:

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GCCCAAGATCATCATTGTGAA, and GACCTTCTCATTTGGGCCATT, were cloned into the pLKO.1 vector for expressing shRNA. The lentivirus production and infection were

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performed as described previously [39].

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Western blot analysis - Western blot analysis was performed as described previously [40]. Briefly,

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30 g of cell lysates per lane were subjected to electrophoresis on 8 or 10% SDS polyacrylamide gels, transferred to an Immobilon PVDF membrane, blocked with 5% milk in TBST, and incubated with the primary antibody (anti-LC3, 1:1000 dilution, Novus Biologicals; antiGAPDH, 1:3000 dilution, Sigma-Aldrich; anti-phospho-CAMK2A, 1:1000 dilution, Santa Cruz Biotechnology). After washing with TBST, the blots were probed with a secondary antibody (1:3000 dilution) for detection by chemiluminescence. Ca2+ measurement- Cytosolic Ca2+ in PC3 cells was performed as described previously [41]. Briefly, cells in 24-well plates were labeled with 4 µM Fura-2 AM in HBSS at room temperature for 20 min, washed, and incubated in regular HBSS (containing 2 mM Ca2+)or Ca2+ free HBSS at room temperature for another 10 min. Thereafter, cells were then put on the stage of an Olympus inverted epifluorescence microscope and fluorescence images were measured by alternate excitation at 340 nm and 380 nm with emission set at 510 nm, and analyzed by a Cell R imaging 13

ACCEPTED MANUSCRIPT software. Colony formation assay- The colony formation assay of PC3 cells was performed as described

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previously [42]. Briefly, PC3 cells were seeded as 1000 cells per well in 6-well plates and

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cultured for 14 days with or without H2O2 treatment. Plates were then washed twice with PBS and fixed with PFA. The colonies were stained with 0.05% crystal violet. The images of each

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well were captured.

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Mitochondrial membrane potential measurement by TMRM- Mitochondria membrane potential indicator Tetramethylrhodamine, Methyl Ester (TMRM, Invitrogen T-668) was dissolved in

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DMSO at a stock concentration of 0.5 mM, and was stored at -20˚C. HeLa or PC3 cells were plated on the 18mm coverslips in a 12-well plate one day before experiment, and were loaded

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with 5 μM TMRM for about 20 min on ice in the dark. The RFP signal of TMRM was excited by 555 nm and collected by the LSM 510 software. The changes of fluorescence intensities were

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processed by Zen 2008 software (Carl Zeiss). Mitochondria morphology- Mitochondria morphology was detected by transfecting a DsRedMito construct into cells. Briefly, HeLa or PC3 cells were plated on the 18mm coverslips in a 12well plate one day before experiment and were transfected with DsRed-Mito plasmid by Lipofectamine 2000. 24 hours after transfection, the mitochondria morphology was imaged in a 63×oil objective (NA 1.4) of Carl Zeiss LSM 510 Meta inverted microscope equipped with Camera Axiocam. Indicated drugs were added to chamber 30 seconds after the first picture. RFP signal was excited by Argon laser at 555 nm and collected with LSM 510 software. The data were processed with Zen 2008 software (Carl Zeiss). Immunocytochemistry- Immunohistochemistry was performed as described previously

[43].

Briefly, cells on cover glasses were fixed with 4% paraformaldehyde, permeabilized with PBS

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ACCEPTED MANUSCRIPT containing 0.1% Triton X-100, blocked with 1% BSA, and incubated with anti-Oxidized-CaMKII (1:1000 dilution, Merck Millipore), followed by secondary antibody (Alexa Fluor® 488 Goat

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Anti-Mouse IgG, A11008, Life Technologies, 1:500 dilution) incubation. DAPI was used to stain

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the nuclei. Cells were imaged using a Zeiss LSM 880 Laser Scanning Microscope.

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Cell viability analysis- Cell viability was assessed by TUNEL assay or MTT assay as described previously [20]. TUNEL assay was performed by following the manufacture’s instruction

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(DeadEnd™ Fluorometric TUNEL System, Promega). For the MTT assay, cells in 96-well plates

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were incubated with 20 μl MTT (5 mg/ml)/per well, and solubilized by 150 μl DMSO/per well. The absorption values were measured by using a Tecan infinite M200 microplate reader using an

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absorbance of 570 nm and a reference of 630 nm.

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Intracellular ROS detection- Intracellular ROS level of HeLa or PC3 cells was assessed by using flow cytometry to analysis the fluorescent intensity of CM-H2DCFDA (Invitrogen) stained cells.

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Briefly, cells were collected, washed twice with DMEM, and incubated with 5 M CMH2DCFDA at 37 °C for 30 min. After incubation, cells were washed twice again with DMEM and analyzed by BD FACSAria I Cell Sorter to determine the fluorescence intensity at excitation and emission wavelengths of 488 nm and 530 nm, respectively. Statistical analysis- Data were presented as mean ± S.E. The statistical significance of differences was calculated by unpaired student's t-test. P < 0.05 was considered as significance.

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ACCEPTED MANUSCRIPT ACKNOWLEDGEMENT We thank members of Yue lab for advice on the manuscript. This work was supported by Hong

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Kong Research Grant Council (RGC) grants (785213 and 17126614), ITS/261/14, CAS-

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Croucher Funding Scheme, Guangdong-Hong Kong joint innovation Research Scheme (#2016A050503010), and Shenzhen government research grant (JCYJ20160229165235739) to

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ACCEPTED MANUSCRIPT Figure Legend Figure 1. In PC3 human prostate cancer cells, TRPM2 was required for oxidative stress-

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mediated cell death induction. (A) H2O2 in a concentration dependent manner (0.5 mM, 1 mM, 2 mM and 4 mM, 6 hours) induced intracellular Ca2+ increases in Fura-2 loaded PC3 cells in

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regular HBSS buffer. (B) H2O2 (1 mM) markedly induced cytosolic Ca2+ increases in Fura-2

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loaded PC3 cells in Ca2+ free HBSS buffer, which was markedly inhibited by TRPM2

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knockdown. (C) TRPM2 knockdown markedly decreased H2O2 (0.5 mM, 1 mM, 2 mM and 4 mM, 6 hours) induced cell death as determined by MTT assay. (D) H2O2 (0.5 mM, 6 hours)

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induced apoptosis in control, but not inTRPM2 knockdown, cells as determined by TUNEL assay.

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Figure 2. In PC3 human prostate cancer cells, TRPM2 was required for oxidative stressmediated autophagy inhibition. (A) H2O2 only induced LC3-II level in TRPM2 knockdown, not

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control, cells in a concentration dependent manner. (B) H2O2 (0.5 mM)-induced LC3-II decreased was abolished by the treatment of CLT (10 M), a TRPM2 inhibitor, in PC3 cells. (C) H2O2 (0.5 mM, 6 hours) markedly induced LC3-II red and yellow puncta in TRPM2 knockdown, but not control, cells.

Figure 3. In TRPM2-overexpressing HeLa cells, oxidative stress induced cell death. (A) H2O2 (25 M, 50 M, 75 M, 100 M and 200 M, 6 hours) markedly decreased cell viability in TRPM2-overexpressing, not the control, HeLa cells in a concentration dependent manner as determined by MTT assay. (B) H2O2 (200 M, 6 hours) markedly induced apoptosis in TRPM2overexpressing, not the control, HeLa cells, which was blocked by CLT (10 M) pretreatment, as determined by TUNEL assay. (C) H2O2 (200 M, 6 hours) induced cell death in TRPM2overexpressing, but not in control, HeLa cells, and this effect was significantly inhibited by the 23

ACCEPTED MANUSCRIPT TRPM2 antagonist, CLT (10 M). Dead cells were detected by propidium iodide (PI) staining of non-fixed cells. Quantification of PI positive cells/total cells are expressed as mean ± S.E., n = 3

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(100-150 cells from each independent experiment). *P < 0.05, scale bar = 25 μm.

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Figure 4. Autophagy inhibition sensitized HeLa cells to oxidative stress-induced cell death. (A) H2O2 (50 M, 75 M, 100 M and 200 M, 6 hours) induced LC3-II levels in control HeLa cells,

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but decreased LC3-II in TRPM2-expressing HeLa cells. (B) and (C) H2O2 (200 M, 6 hours)

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markedly decreased the cell viability (B) or induced apoptosis (C) in BAF (10 nM)-treated control HeLa cells, yet BAF had little effects on H2O2-induced toxicity in TRPM2 expressing

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HeLa cells. (D) ATG5 knockdown blocked H2O2 (75 M, 6 hours) induced LC3-II levels in control HeLa cells, but had no effect on the H2O2-mediated decrease in LC3-II in TRPM2-

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expressing HeLa cells. (E) H2O2 (200 M, 6 hours) induced apoptosis in ATG5 knockdown, but not control, HeLa cells. Quantification of TUNEL assay (C and E) is presented as TUNEL

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positive cells/DAPI-stained cells ± S.D., n = 3 (100-150 cells per experiment), *P < 0.05. Scale

Figure 5. Oxidative stress induced intracellular ROS production and triggered mitochondria fragmentation in TRPM2 dependent manner. (A) H2O2 (0.2 mM, 0.5 mM and 1 mM, 30 min) markedly increased intracellular ROS levels in control, not TRPM2 knockdown, PC3 cells. (B) H2O2 (100 M, 200 M and 500 M, 30 min) markedly increased intracellular ROS levels in TRPM2-overexpressing, not control, HeLa cells. (C) and (D) H2O2 (200 M) markedly induced mitochondria fragmentation in control PC3 cells (4 hours) (C) or TRPM2-overexpressing HeLa cells (D). (E) and (F) H2O2 (200 M) markedly decreased mitochondrial membrane potential levels in control PC3 cells (E) or TRPM2-overexpressing HeLa cells (F).

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ACCEPTED MANUSCRIPT Figure 6. Oxidative stress activated CaMKII to induce cell death in PC3 cells. (A) H2O2 did not change the autophosphorylation of CaMKII in PC3 cells. (B) H2O2 (75 M) treatment (30min)

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markedly increased the oxidation of CaMKII in control, not TRPM2 knockdown, PC cells as

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shown by the immunofluorescent staining analysis with an oxidative-specific antibody against CAMK2-(C)M281/282. (C) Treatment of AIP (5 M), an CaMKII inhibitor, abolished H2O2 (0.5

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mM for 6 hours)-mediated LC3-II decrease in PC3 cells. (D) AIP (5 M) treatment mitigated

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H2O2 (0.5 mM for 6 hours)-induced cell death in PC3 cells. (E) Expression of CaMK2AK42R, a

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dominant negative mutant of CaMKII, also inhibited H2O2 (0.5 mM, 1 mM, 2 mM, and 4 mM for 6 hours)-induced cell death in PC3 cells.

Figure 7. CaMKII is required for oxidative stress-induced ROS increase in TRPM2-expressing

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cells. (A) AIP (5 M), an CaMKII inhibitor, treatment significantly inhibited TRPM2-mediated intracellular ROS increases in PC3 cells. (B) KN93 (80 M), an CaMKII inhibitor, or CLT (10

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M), an TRPM2 inhibitor, or NAC (10 mM) treatment mitigated oxidative stress-induced intracellular ROS increase in TRPM2-overexpressing HeLa cells. (C) AIP (5 M) treatment prevented H2O2-induced mitochondrial membrane potential loss in PC3 cells.

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Highlights 1. Oxidative stress activated the TRPM2-Ca2+-CaMKII cascade to inhibit early autophagy induction, which ultimately led to cell death in TRPM2 expressing cancer cells. 2. TRPM2 knockdown switched cells from cell death to autophagy for survival in response to oxidative stress. 3. Oxidative stress activated the TRPM2-CaMKII cascade to further induce intracellular ROS production, which led to mitochondria fragmentation and loss of mitochondria membrane potential.

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