IJC International Journal of Cancer

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1 IJC International Journal of Cancer Blocking mammalian target of rapamycin alleviates bone cancer pain and morphine tolerance via m-opioid receptor Zongming Jiang 1*, Shaoyong Wu 2*, Xiujuan Wu 3, Junfeng Zhong 1, Anqing Lv 1, Jing Jiao 4 and Zhonghua Chen 1 1 Department of Anesthesiology, Shaoxing People s Hospital (Shaoxing Hospital of Zhejiang University), Shaoxing, Zhejiang , China 2 Department of Anesthesiology, Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangdong , China 3 Department of Nephrology, Shaoxing People s Hospital (Shaoxing Hospital of Zhejiang University), Shaoxing, Zhejiang , China 4 Department of Anesthesiology, Shanghai Obstetrics and Gynecology Hospital, Fudan University, Shanghai , China The current study was to examine the underlying mechanisms responsible for the role of mammalian target of rapamycin (mtor) in regulating bone cancer-evoked pain and the tolerance of systemic morphine. Breast sarcocarcinoma Walker 256 cells were implanted into the tibia bone cavity of rats and this evoked significant mechanical and thermal hyperalgesia. Our results showed that the protein expression of p-mtor, mtor-mediated phosphorylation of 4E binding protein 4 (4E-BP1), p70 ribosomal S6 protein kinase 1 (S6K1) as well as phosphatidylinositide 3-kinase (p-pi3k) pathways were amplified in the superficial dorsal horn of the spinal cord of bone cancer rats compared with control rats. Blocking spinal mtor by using rapamycin significantly attenuated activities of PI3K signaling pathways as well as mechanical and thermal hyperalgesia. Additionally, rapamycin enhanced attenuations of protein kinase CE (PKCE)/protein kinase A (PKA) induced by morphine and further extended analgesia of morphine via m-opioid receptor (MOR). Our data for the first time revealed specific signaling pathways leading to bone cancer pain, including the activation of mtor and PI3K and downstream PKCE/PKA, and resultant sensitization of MOR. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of bone cancer pain often observed in clinics. Pain is one of the most common and distressing symptoms suffered by patients with progression of cancer. 1,2 Cancer pain mainly arises from a tumor compressing or infiltrating tissue; from nerve and other changes caused by a hormone imbalance or immune response and so forth. 3 Of note, cancerous cells can originate in a number of different tissues such as prostate; breast and lung. Many types of cancers have a propensity to metastasize to the bone microenvironment. 3,4 Tumor burden within the bone causes excruciating breakthrough pain with properties of ongoing pain that is inadequately managed with current analgesics. Treatment options Key words: bone cancer, neuropathic pain, mtor, morphine tolerance Abbreviations: DRG: dorsal root ganglion; MOR: m: opioid receptor; mtor: mammalian target of rapamycin; PKA: protein kinases A; PKC: protein kinases C; PWL: paw withdrawal latency; PWT: paw withdrawal threshold *Z.J. and S.W. contributed equally to this work. DOI: /ijc History: Received 4 Apr 2015; Accepted 20 Oct 2015; Online 13 Nov 2015 Correspondence to: Dr. Zhonghua Chen, Department of Anesthesiology, Shaoxing People s Hospital (Shaoxing Hospital of Zhejiang University), 568 North Zhongxing Road, Shaoxing, Zhejiang , China, Tel.: , Fax: , zhonghuachen64@163.com for bone cancer pain have been limited, partly due to our poor understanding of the underlying mechanisms responsible for pain. Although opioids such as morphine are used for the treatment of advanced bone cancer pain, an increasing dose of morphine are required. 5 Given that the side effects caused by a higher dose of morphine, development of a novel strategy to enhance the analgesic effects of morphine to control bone cancer pain and further to reduce its side effects also becomes an important issue for treatment and management of patients with advanced bone cancer. Mammalian target of rapamycin (mtor) is a serine threonine protein kinase. Activation of mtor, in particular, mtor complex 1 (mtorc1) that is more sensitive to rapamycin, leads to promotion of the phosphorylation of downstream effectors, such as p70 ribosomal S6 protein kinase (p70s6k) and this further governs mrna translation. 6 The mtorc1 is well known for its critical roles in the regulation of protein synthesis and growth and further the compelling evidence supports the widespread dysregulation of mtor and its downstream pathways in human cancer. 7 In particular, bone is the most common site of metastases for breast carcinoma 8 and mtor signaling plays an important role in in regulating osteoclast survival in bone cancer. 9,10 Formation of osteolytic lesions by tumors metastasizing to bone depends on the activation of bone-resorbing osteoclasts. 11 Metastatic cells arriving at the bone site is likely to obstruct osteoblast

2 2014 mtor and bone cancer pain What s new? Bone metastases cause localized breakthrough pain, but they can also increase a patient s overall sensitivity to pain. Treatment options for bone-cancer pain have been limited, in part because the underlying mechanisms of pain are poorly understood. In this study in mice, the authors found that when mtor signaling pathways in the spine were blocked with rapamycin, pain sensitivity decreased. In addition, morphine tolerance was reduced. The study also identified specific molecular signaling pathways, which suggest potential new therapeutic targets for bone-cancer pain. maturation. 12,13 This leads to increased expression by immature osteoblasts of osteoclastogenic substances and further induces formation and activation of osteoclasts. Breast carcinoma cells can directly evoke osteoclastogenesis independent of supporting bone cells. 14 Pathologically activated osteoclasts in turn resorb the inorganic bone matrix destroying the normal bone architecture, compromising bone integrity and liberating tumor-supportive growth factors stored in the bone such as tumor growth factor that may further stimulate tumor growth. 15 During this pathological processing of tumor growth, mtor signaling pathways are activated and amplified 9 and likely to cause secondary complications of bone metastases such as pain. As a key region of pain regulation, 16,17 the spinal cord dorsal horn is likely engaged in the effects of mtor on bone cancer-induced pain. Thus, in this report we implanted breast sarcocarcinoma Walker 256 cells into the tibia bone cavity of rats. We suspected that mtor in the dorsal horm of the spinal cord are likely changed by implanting tumor cells into the tibia bone cavity. We also suspected that mtor is likely an important player for the induction and maintenance of bone induced-cancer pain. In addition, prior studies have strongly supported the notion that mtor plays an important role in the modulation of long-term neuronal plasticity. 18,19 Specifically, mtor and its downstream effectors have been identified in dorsal root ganglion and spinal cord dorsal horn as major regions in the process of nociception and recent studies further suggest that mtor contributes to transmission and modulation of nociceptive information. 20 For example, intrathecal administration of rapamycin, a specific inhibitor of mtor, produces antinociception in models of nerve injury and inflammation Local perfusion of rapamycin into the spinal cord significantly attenuates formalin-induced neuronal hyperexcitability in the dorsal horn. 23 These findings indicate that mtor and its downstream effectors are likely activated and play an important role in the development of spinal central sensitization under persistent pain conditions. Therefore, we hypothesized that bone cancer increases the protein expression of mtor and its downstream PI3K pathways in the superficial dorsal horn, resulting in mechanical and thermal hypersensitivity. We speculated that blocking spinal mtor by intrathecal injection of rapamycin would attenuate activities of phosphatidylinositide 3-kinase (PI3K) pathways thereby leading to a reduction in mechanical and thermal sensitivity. We further hypothesized that mtor is engaged in regulating the tolerance of systemic morphine. We speculated that blocking spinal mtor would attenuate expression of protein kinase CE (PKCE)/protein kinase A (PKA) and thereby blunt morphine tolerance via a m-opioid receptor (MOR) mechanism. Material and Methods Animal A total of 136 Wistar rats weighing g were obtained from the Institutional Center of Experimental Animal. All animal protocols were approved by the Animal Care and Use Committee of this intuition and were carried out in accordance with the guidelines of the International Association for the Study of Pain. Cell preparation Wister rat breast sarcocarcinoma Walker 256 cells were prepared as described previously. 24,25 Briefly, Walker 256 cells ( in 0.5 ml) were injected into the abdominal cavity of the rats. Seven to 14 days later, the produced ascites were collected and centrifuged. The cells in the ascites were washed three times with 10 ml D-Hank s solution and diluted to a final concentration of cells/ml. The cells were then kept on ice before being used. A model of bone cancer pain The bone cancer pain model was established by inoculating Walker 256 cells to the tibia of the rats. 24,25 Briefly, the rats were anesthetized with sodium pentobarbital (45 mg/kg, i.p.) and the lower one-third of the tibia was exposed. Each of rats was injected with Walker 256 cells in 5 ll Hank s solution into the right tibia of the hind paw. Rats that underwent the same surgical procedures and received vehicle injection served as the sham controls. Intrathecal catheter for administration of drugs Rats were anesthetized by sodium pentobarbital (45 mg/kg, i.p.) in order to implant intrathecal catheter for administration of drugs. 24 In each experiment, the intrathecal tubing was connected to an osmotic mini pump (Alzet) to intrathecally deliver saline as control and antagonists to mtor (rapamycin, obtained from Selleckchem) and to MOR (CTOP, obtained from Tocris) and inhibitor of PI3K (LY294002, obtained from Sigma-Aldrich). Rapamycin and LY were dissolved in 50% DMSO to obtain stock

3 Jiang et al Figure 1. Development of mechanical and thermal hyperalgesia began within 7 days after implantation of Walker 256 cells into the tibial canal of rats and lasted for the rest of days tested. Left panel: Intrathecal administration of rapamycin increased PWT and PWL in bone cancer rats. *p < 0.05 versus saline (vehicle control) and 1 mg of rapamycin. N 5 12 in controls and n in three groups with different dosages. Right panel: Intrathecal injection of LY (PI3K inhibitor) increased PWT and PWL in bone cancer rats. N 5 10 in saline control and n in injection of LY with three different dosages. *p < 0.05 versus saline control and 1 mm of LY solution and dissolved in saline to obtain target concentrations when they were used for infusion. The drugs were constantly delivered at 1 ll/hr for 14 days after an initial activation period of 4 hrs inside the animal. Behavioral test To quantify the mechanical sensitivity of the hindpaw, rats were placed in individual plastic boxes and allowed to acclimate for > 30 min. Mechanical paw withdrawal threshold (PWT) of rat hind paw in response to the stimulation of von Frey filaments was determined. The tactile stimulus producing a 50% likelihood of withdrawal was determined using the up-down method. 26 Each trial was repeated two times at approximately 2 min intervals. The mean value was used as the force produced a withdrawal response. To determine thermal hyperalgesia, rat paw withdrawal latency (PWL) to a radiant heat was measured. 24,25 Rats were placed individually in plastic cages on an elevated glass platform and allowed for 30 min acclimation. Each hind paw received three stimuli with a 10 min interval, and the mean of the three withdrawal latencies was defined as PWL. The heat was maintained at a constant intensity. To prevent tissue damage, the cut-off latency was set at 20 sec. Western blot analysis The rats were euthanized and the superficial dorsal horn (lumbar 4 6) tissues were dissected under an anatomical microscope. The tissues were then homogenized, centrifuged and incubated with streptavidin beads. Streptavidin was used to minimize steric hindrance of the subsequent binding of antigen to the antibody and optimize results of Western blot. The beads were washed and precipitated by centrifugation and sample buffer was added to the collected beads. Beads were pelleted again by centrifugation to obtain supernatant. The supernatant was then diluted to the same volume and applied to SDS-PAGE. Membranes were incubated with the

4 2016 mtor and bone cancer pain Figure 2. Averaged data and typical bands (a): Showing that p-mtor, p-s6k1 and p-4e-bp1 in the dorsal horn of the spinal cord were upregulated in bone cancer rats (n ). *p < 0.05 versus control rats (n ). (a): Also, showing that mtor, S6K1 and 4E-BP1 in the dorsal horn of the spinal cord were upregulated in bone cancer rats (n ). The ratio of p-mtor, p-s6k1 and p-4e-bp1 levels versus mtor, S6K1 and 4E-BP1 levels was significantly increased in bone cancer rats, respectively. *p < 0.05 versus control rats (n ). Averaged data and typical bands (b, c, d): Expression of p-pi3k, PI3K, p-s6k1, S6K1, the ratio of p-s6k1 and S6K1, PKCE and PKA were amplified in the dorsal horn of bone cancer rats with saline (vehicle) (n ) as compared with control rats (n 5 6 8). Increases of p- PI3K, PI3K, p-s6k1, S6K1, the ratio of p-s6k1 and S6K1, PKCE and PKA were attenuated after rapamycin was infused via an osmatic mini pump. *p < 0.05 versus control animals and bone cancer animals infused with rapamycin (n in each group). rabbit anti-p-mtor (ab84400)/p-s6k1(ab60948)/p-4e-bp1 (ab47365) antibodies; rabbit anti-mtor (ab2732)/ S6K1(ab32359)/4E-BP1 (ab2606) antibodies; mouse anti- PI3K p85 (ab86714); rabbit anti-p-pi3k p85 (ab182651) and anti-pkce (ab63387)/pka (ab5815) antibodies, respectively. All these primary antibodies were purchased from the Abcam Company; and goat anti-rabbit and anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology. Immunoreactive proteins were detected by enhanced chemiluminescence. The membrane was also processed to detect b-

5 Jiang et al Figure 3. (a and b): Showing effects of co-administration of a sub-dose of rapamycin and morphine on bone cancer-induced hyperalgesia. A sub-dose of rapamycin did not significantly increased PWT and PWL (n 5 8), but this significantly amplified effects evoked by systemic morphine on PWT and PWL (n 5 10). Importantly, rapamycin also extended the length of analgesia induced by morphine. Note that a preinfusion of CTOP attenuated the effects of rapamycin on enhancement of analgesia of morphine (n 5 12). *p < 0.05 versus controls (n 5 8), morphine injection (n 5 10), and morphine with prior CTOP and rapamycin. (c and d): Systemic injection of morphine attenuated PKCE and PKA expression as well as p-s6k1 in the dorsal horn of bone cancer rats (n 5 10). A sub-dose of rapamycin significantly amplified the effects of morphine on the expression of PKCE, PKA and p-s6k1 (n 5 8). *p < 0.05, bone cancer rats with saline (vehicle) (n 5 10) versus control animals (n 5 6) and bone cancer animals infused with morphine, morphine plus rapamycin. p < 0.05 indicated, morphine injection versus morphine plus rapamycin. There was no a significant difference between saline control and morphine plus rapamycin with pretreatment of CTOP in cancer rats. actin for equal loading. The optical density of protein bands was first analyzed using the NIH Scion image Software, and values for densities of immunoreactive bands/b-actin band densities from the same lane were determined. Each of the values was then normalized to a control sample. Statistical analysis All data were analyzed using a two-way repeated-measure ANOVA. Values were presented as means 6 SEM. For all analyses, differences were considered significant at p < All statistical analyses were performed by using SPSS 13.0 (SPSS).

6 2018 mtor and bone cancer pain Results Consistent with previous findings, no significant mechanical and thermal hyperalgesia were observed in control rats. 24,25 Significant mechanical and thermal hyperalgesia were developed within a week after implantation of Walker 256 cells into the tibial canal of rats and lasted for 4 weeks. Accordingly, in the current report the rats were subjected to all the experiments before inoculation of cancer cells and 3 days to two weeks after inoculation. mtor engaged in hyperalgesia Mechanical and thermal sensitivities were examined before inoculation and 3, 7, 10 and 14 days after intrathecal rapamycin (1, 5, 10 mg, n in each group). Figure 1 left panel showed that rapamycin at dosages of 5 and 10 mg significantly attenuated bone cancer-induced mechanical and thermal hyperalgesiain in dosage-dependent and timedependent manners (p < 0.05 vs. vehicle control, n 5 12; and 1 mg of rapamycin). The inhibitory effects of rapamycin on mechanical and thermal hyperalgesia began 7 days after inoculation. PI3K signaling pathways in mtor mediating hyperalgesia Specific PI3K inhibitor LY (1, 5, 10 lm) was intrathecally infused, respectively. Figure 1 right panel demonstrated that LY had significant attenuating effects on bone cancer-induced mechanical and thermal hyperalgesia in dosage and time-dependent manners. At a dose of 1 lm, LY has no effects on hyperalgesia 7 days after inoculation (p > 0.05, 1 lm of LY vs. vehicle). At doses of 5 and 10 lm, the effects of LY on hyperalgesia were observed (p < 0.05, LY vs. vehicle, n for LY and n 5 10 for vehicle). Figure 2a showed the protein expression of p-mtor, p- S6K1 and p-4e-bp1 as well as mtor, S6K1 and 4E-BP1 in control rats and rats injected with Walker 256 cells for two weeks. Bone cancer significantly increased the protein levels of p-mtor and mtor-mediated p-s6k1 and p-4e-bp1 in the superficial dorsal horn as compared with control rats (p < 0.05, cancer rats vs. control rats, n in each group). Also, the ratio of p-mtor, p-s6k1 and p-4e-bp1 levels versus total protein of mtor, S6K1 and 4E-BP1 levels was significantly increased in bone cancer rats. We also examined the effects of blocking mtor on expression of p- PI3K, PI3K, PKCe and PKA. Figures 2b22d demonstrated that the protein expression of p-pi3k, PI3K, PKCe and PKA was significantly increased in bone cancer rats (n ) as compared with control rats (n 5 6-8). Note that the ratio of p-pi3k and PI3K was also significantly increased in bone cancer rats. When rapamycin was infused into the spinal cord of cancer rats via an osmotic mini pump, the amplified activities of p-pi3k, PI3K, the ratio of p-pi3k and PI3K, PKCe and PKA evoked by bone cancer were significantly attenuated (n in each group). In addition, Figures 2b22d showed that mtor signaling pathway was inhibited by rapamycin because as an indicator of mtor expression, p-s6k1, the ratio of p-s6k1 and S6K1 (b) and p- S6K1 (c, d), were significantly decreased after application of rapamycin. Interaction of mtor and morphine in modulating hyperalgesia We further examined the effects of co-administration of a sub-dose (1 mg) of rapamycin and morphine on bone cancer-induced hyperalgesia. We selected 1 mg of rapamycin because this sub-dose failed to alter PWT and PWL per se as shown in Figure 1. This allowed us to examine its combination effects with morphine on pain responses. Figures 3a and 3b) demonstrated that a sub-dose of rapamycin failed to increase PWT and PWL (n 5 12), but this significantly amplified effects evoked by systemic morphine (2 mg/kg i.p. daily) on PWT and PWL (n 5 10). Of note, rapamycin extended the length of analgesia induced by morphine. i.e. increases in PWT and PWL lasted for > 10 days. In contrast, the analgesia evoked by this dose of morphine alone was < 7 days (n 5 8). In addition, infusion of MOR antagonist, CTOP (15 mg), 27 significantly attenuated the effects of rapamycin on enhancement of analgesia of morphine (n 5 12). We also examined the mechanisms by which mtor contributes to the effects of morphine on bone cancer pain. Figures 3c and 3d demonstrated that systemic injection of morphine (2 mg/kg i.p. daily) significantly attenuated PKCE and PKA expression in the dorsal horn in bone cancer rats (n 5 10). Figures 3c and 3d further showed that a sub-dose of rapamycin (1 mg) significantly amplified the attenuating effects of morphine on the expression of PKCE and PKA (n 5 8). Note that a sub-dose of rapamycin failed to alter PKCE and PKA per se. There was no a significant difference between saline control and morphine plus rapamycin with pretreatment of CTOP in cancer rats. In addition, Figures 3c and 3d showed that expression of p-s6k1was significantly decreased by morphine and co-injection of morphine and rapamycin (1 mg), suggesting that mtor signaling pathway is inhibited by rapamycin and mtor is engaged in the effects of morphine. Discussion There are two distinct mtor forms of protein complexes, mtor complex 1 (mtorc1) and mtorc2. In general, mtorc1 is composed of raptor, mlst8, and mtor, and is known to gate translation of most proteins by phosphorylation of specific downstream effectors including, 4E-BPs and p70 ribosomal S6Ks. 24 mtor, S6K1 and 4E-BP1 are expressed in the mammalian nervous system, particularly in spinal cord dorsal horn. 19,24 Because the superficial dorsal horn is the first synaptic site from peripheral afferent nerves to the central nervous system 28,29 and plays an important role in modulating pain and morphine tolerance, 16,17 in this

7 Jiang et al study we determined the role played by mtor in the superficial dorsal horn in regulating mechanical and thermal hyperalgesia as well as morphine-induced adaptive changes that occur in protein translation and contribute to the development and maintenance of morphine-induced tolerance. We consistently observed development of mechanical and thermal hyperalgesia in bone cancer rats (Fig. 1). We also demonstrated that expression of p-mtor, p-s6k1 and p-4e- BP1 in the superficial dorsal horn of bone cancer rats was upregulated, and mtor antagonist rapamycin injected the dorsal horn attenuated mechanical and thermal hyperalgesia evoked by bone cancer (Fig. 1). The PI3K/Akt pathway is an intracellular signaling pathway in regulating the cell cycle. This important mechanism is directly related to cellular quiescence, proliferation, cancer and longevity. PI3K can phosphorylate and activate Akt in the plasma membrane. 30 The, Akt leads to several downstream effects which alters transcription of p70 ribosomal S6K1 or 4E-BP1 and activating camp response elementbinding protein (CREB) and inhibition of p27 and so forth. 6,7,16,30 Our study demonstrated expression of p-pi3k in the dorsal horn of bone cancer rats was upregulated, and intrathecal injection of rapamycin and PI3K inhibitor attenuated mechanical and thermal hyperalgesia evoked by bone cancer (Fig. 1). This suggests that PI3K is necessary to play a regulatory role in mediating the effects of mtor on bone cancer evoked-pain responses. Blocking mtor also attenuated p-pi3k expression (Fig. 2). It is assumed that rapamycin may play a role in regulating the negative feedback loops emanating from S6K1 to PI3K signaling pathway. This has been shown to be relevant in many cancer cell lines. 31 Insulin receptor substrate 4 (IRS1) is one of the major substrates of the insulin receptor kinase, which contains multiple tyrosine phosphorylation motifs to play a role as docking sites for SH2-domain in mediating the metabolic and growth-promoting functions of insulin. 32,33 In general, the PKC and mtor pathways mediate phosphorylation of IRS1 at Ser612 and Ser636/639; whereas PKCu specifically mediates phosphorylation of IRS1 at Ser1101, 34,35 resulting in an inhibition of insulin signaling in the cell. Nonetheless, our results showed that p-mtor and PKC were increased in the dorsal horn of bone-cancer rats in addition to increases of S6K1, PI3K and PKA pathways, likely leading to IRS1 activation. The evidence supports the idea that rapamycin may activate PI3K pathway via S6K1-IRS1 feed-back regulatory mechanisms. 31 Although it still needs more mechanistic data to support this notion, this raises an important issue if rapamycin somehow influences the PI3K signaling pathway and mtorc1 activation. The mtor signaling pathways are important mechanisms responsible for osteoclast survival in bone cancer. 9,10 Rapamycin is a classical inhibitor of mtor activity. Prior studies have demonstrated that rapamycin plays a beneficial role in improving osteolysis and survival in animal models of cancer. 36 In our current study, we have observed that mtor expression in the dorsal horn was increased after implantation of breast sarcocarcinoma Walker 256 cells into the tibia bone cavity of rats. Also, PI3K pathway in the dorsal horn was increased and administration of rapamycin significantly attenuated PI3K expression. In addition to expression of mtor and PI3K, we further determined activities of mtor and PI3K. Our results showed that p- mtor and p-pi3k were significantly enhanced in the dorsal horn of bone-cancer rats. Importantly, rapamycin significantly attenuated p-pi3k activity. Moreover, p-s6k1 and p- 4E-BP1 were increased in the dorsal horn after implantation of breast cells. Overall, data of our current study support the role played by the mtor signaling pathways in regulating mechanical and thermal hypersensitivity observed in bone-cancer. A prior study has demonstrated that MOR expression is down-regulated in sensory neurons of bone cancer rats. 37 As G-protein-coupled receptors, MOR activates downstream PKC and PKA pathways and alters the levels of camp, all of which are involved in morphine tolerance. 38 Accordingly, we examined the spinal mechanisms by which mtor is likely responsible for morphine tolerance in modulating bone cancer pain. We showed that blocking mtor decreased PKCE and PKA expression in the dorsal horn of cancer rats (Fig. 2). Also, systemic injection of morphine attenuated PKCE and PKA expression in the superficial dorsal horn of cancer rats and a sub-dose of rapamycin significantly amplified the effects of morphine on the expression of PKCE and PKA (Fig. 3). Likewise, we further showed that a sub-dose of rapamycin amplified the amplitude of anti-nociception evoked by systemic morphine, but also extended the length of antinociception of morphine (Fig. 3). We suggest that blocking mtor alleviated morphine tolerance likely via a MOR mechanism because the effects of rapamycin were significantly decreased after the prior administration of a specific MOR antagonist (Fig. 3). Collectively, we suggest that spinal mtor contributes to morphine tolerance in modulating bone cancer pain likely via engagement of PKCE and PKA linked to activation of MOR. Interestingly, a recent study using normal animals suggested that spinal mtor and its downstream effectors contribute to the development and maintenance of chronic morphine tolerance and morphine-induced hyperalgesia. 39 The effects of mtor are activated through a l opioid receptor triggered PI3K/Akt pathway in dorsal horn neurons during chronic morphine exposure. Nonetheless, it was observed that spinal mtor pathway is activated in animals with bone cancer-induced pain and rapamycin attenuates cancerinduced mechanical allodynia and thermal hyperalgesia during both development and maintenance of cancer-related pain. 40 Our current findings are consistent with the data reported by this previous study. Our data further suggest that

8 2020 mtor and bone cancer pain mtor is engaged in morphine tolerance via PKCE/PKA and MOR mechanisms. In conclusion, in bone cancer, spinal p-mtor and mtor-mediated p-s6k1 and p-4e-bp1 as well as PI3K are upregulated, which results in mechanical and thermal hypersensitivity. Blocking mtor and PI3K attenuates bone cancer pain. In addition, blocking mtor blunts morphine tolerance via PKCE/PKA and MOR mechanisms. Conflict of Interest None References 1. Magdi H, Zylicz Z, eds. Cancer Pain. London: Springer, vii & van den Beuken-van Everdingen MH, de Rijke JM, Kessels AG, et al. Prevalence of pain in patients with cancer: a systematic review of the past 40 years. Ann Oncol 2007;18: Urch CE, Suzuki R. Pathophysiology of somatic, visceral, and neuropathic cancer pain. In: Sykes N, Bennett MI, Yuan C-S, eds. Clinical pain management: cancer pain. London: Hodder Arnold, Mantyh P. Bone cancer pain: causes, consequences, and therapeutic opportunities. Pain 2013;154 Suppl 1:S Schug SA, Auret K. 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