Our data show that (a) acute and prolonged inhibition of p38 MAPK blocks non-evoked pain behaviors as measured by flinching and guarding, but not response thresholds to evoked stimuli; (b) prolonged blockade of p38 MAPK attenuates tumor-induced bone remodeling; and (c) prolonged blockade of p38 MAPK diminishes tumor growth within the bone. These data indicate that components of cancer-induced bone pain that may reflect ongoing pain and evoked hypersensitivity are likely driven by independent mechanisms. These findings suggest that tumor-induced ongoing pain is dependent on p38 MAPK signaling. However, inhibition of p38 MAPK signaling is insufficient to block hypersensitivity to evoked stimuli, which may be dependent on establishment of altered central processing (i.e., central sensitization). Importantly, we demonstrate that chronic inhibition of p38 MAPK failed to attenuate tactile hypersensitivity despite markedly diminished tumor within the bone and bone remodeling, suggesting potential distinction between anti-tumor activity and tactile hypersensitivity.
Previous studies in mice have demonstrated increased phosphorylated p38 MAPK in the DRG and spinal cord dorsal horn neurons ipsilateral to osteosarcoma treated femurs  and in spinal microglia , indicating potential peripheral and central actions of p38 MAPK in cancer-induced bone pain. Activation of p38 MAPK has been implicated in numerous translational and transcriptional responses implicated in peripheral and spinal sensitization initiated by inflammatory and neuropathic injury [16, 17, 29–32]. Previous studies have demonstrated that p38 MAPK mediates increased transcription of nociceptive peptides, such as SP and CGRP, within the DRG [29, 33]. Further, p38 MAPK mediates injury-induced increased production of TRPV1 and TRPA1 within the DRG [30, 34], and trafficking of the TRPV1 channel to the periphery [30, 35, 36]. Notably, p38 MAPK drives post-translational changes implicated in pain such as phosphorylation of the TRPV1 channel, resulting in reduced activation threshold and potentiated capsaicin- or proton-evoked responses . Phosphorylation of the Nav1.8 channel increases current density that can increase neuronal excitability under pathological conditions . Thus, the effects of acute administration of SB 203580 may be from inhibition of the direct activation/phosphorylation of pro-nociceptive channels. As tumor- and osteoclast- induced acidosis of the tumor microenvironment are implicated in tumor-induced bone pain , increases in acid sensing channels within the bone, decreased activation thresholds within the tumor microenvironment, and increased neuronal excitability, likely result in increased activation of sensory fibers by factors within the tumor microenvironment. Supporting this, blockade of the TRPV1 receptor attenuated osteosarcoma-induced flinching and guarding behavior [39–42]. Thus, prolonged administration of the p38 MAPK inhibitor may diminish cancer-induced ongoing pain behaviors by multiple mechanisms associated with up-regulation and trafficking of pronociceptive signaling channels (e.g., TRPV1) to the periphery.
Within the tumor microenvironment, multiple disease related factors sensitize and directly drive nociceptive afferent fibers [3, 43]. In addition to tumor and osteoclast induced acidosis, mechanical factors, such as compression and damage to sensory fibers innervating the bone by tumor growth, or mechanical stress from destabilization of the bone structure due to tumor induced bone remodeling may drive afferent activity [3, 43]. Such signal may be amplified due to the phosphorylation of sodium channels . Moreover, pronociceptive inflammatory mediators including endothelins, ATP, prostaglandins, growth factors and pronociceptive cytokines are released from the tumor, tumor-induced bone resorption, and infiltrating immune cells such as tumor associated macrophages [3, 27, 44–48]. These factors are implicated in sensitization and direct activation of sensory fibers [3, 27, 44]. Moreover, p38MAPK has been implicated in release of many of these factors including release of ATP from tumor associated macrophages  and release of cytokines [22, 23, 49, 50]. More recently, systemic administration of a selective p38α MAPK inhibitor has been shown to inhibit lipopolysaccharide (LPS)-induced elevation of blood levels of tumor necrosis factor-alpha (TNFα) in humans . Thus, the blockade of tumor-induced flinching and guarding behaviors may be due to diminished release of factors within the tumor microenvironment that sensitize primary afferent fibers, thereby lowering thresholds for direct activation by factors within the bone microenvironment (e.g. acid, ATP), and directly reducing factors (e.g. ATP) that may directly drive afferent input from the tumor microenvironment.
In addition to directly inhibiting release of pronociceptive factors from the tumor and associated immune cells, administration of the p38 MAPK inhibitor across 7 days also demonstrated disease modifying effects. Painful bone metastases in humans, such as prostate and breast cancer, often have osteolytic and osteoblastic lesions [52, 53]. Our model shows tumor-induced osteolytic lesions by day 10 followed by osteoblastic activity. Prolonged SB203580 treatment completely eliminated the abnormal osteoblastic structures observed in the vehicle-treated bones by day 13 post-tumor implantation. In addition, radiographic analyses of bones showed reduced incidences of spontaneous bone fractures in drug treated bones as compared to those treated with the vehicle. Thus, blockade of tumor-induced bone remodeling likely contributed to the blockade of pain behaviors in cancer bearing mice treated with prolonged administration of the p38 MAPK inhibitor. In addition, histology of bones collected at the end our study show significant tumor reduction with prolonged drug treatment. Moreover, administration of the p38 MAPK inhibitor to cultured 66.1 cells diminished viability, indicating a role of p38 MAPK in tumor growth. This is consistent with multiple reports implicating p38 MAPK in proliferation, invasion and migration of a variety of malignancies including breast cancer [54–58]. These tumorigenic effects of p38 MAPK may be mediated by modulation of transcription factors (e.g. NF-κB [59, 60]) and regulation of anti-apoptotic inflammatory signals (e.g. interleukin-6 (IL-6) ). SB203580 has been demonstrated to inhibit cyclooxygenase-1 and -2 (COX-1 and 2) . This is notable as COX-2 inhibitors have been demonstrated to diminish tumor growth within bone, bone loss and bone pain in a mouse model of sarcoma-induced bone pain . We demonstrate that treatment with 15 mg/kg of SB203580 restricted tumor growth to the shaft of the bone, with absence of tumor growth into the epiphyseal plate region. At a higher dose, 30 mg/kg, only small pockets of tumor were seen. We note that this is in contrast with previous reports that administration of a p38 MAPK inhibitor across 9-11 days failed to inhibit growth of osteosarcoma cells within the mouse femur . These discrepant results may be due to numerous factors including route of administration (food chow as opposed to twice daily i.p. injections), and cell lines used (osteosarcoma vs. murine cell line 66.1 mammary adenocarcinoma cells). Our data suggest that diminished tumor burden and tumor-induced bone remodeling may be key factors contributing to blockade of tumor-induced flinching and guarding pain behaviors.
Consistent with previous findings in the osteosarcoma pain model , p38 MAPK inhibition failed to block tactile hypersensitivity. One potential explanation for the divergent effects of p38 MAPK inhibition on flinching and guarding as opposed to tactile hypersensitivity is the site of action. We propose that activation of afferent fibers innervating the tumor microenvironment drives cancer-induced ongoing pain whereas central mechanisms mediate tactile hypersensitivity. Tactile hypersensitivity is a measure of referred pain, which likely reflects hypersensitivity to touch stimuli, which is mediated by central sensitization . Previous studies have demonstrated phosphorylation of p38 MAPK in the spinal cord across several injury states such as inflammation-induced pain [17, 64] neuropathic pain [16, 31, 65], spinal injury  incision , and osteosarcoma induced bone pain [27, 28]. Several studies have demonstrated that spinal administration of the p38 MAPK inhibitor starting before or at the time of injury blocks evoked pain (tactile and thermal hypersensitivity) [18, 68–70]. In contrast, others have demonstrated that spinal administration of p38 MAPK inhibitors after the pain state is established failed to reverse thermal or tactile hypersensitivity [68, 71, 72]. Thus, although p38 MAPK is implicated in initiation of spinal sensitization, it does not appear to be a factor in maintaining central sensitization and the associated thermal or tactile hypersensitivity. We propose that growth of the tumor within the bone and the associated afferent drive may have established central sensitization required to maintain tactile hypersensitivity prior to drug delivery, as mice demonstrated flinching, guarding, impaired limb use, and tactile allodynia within 7 days, prior to drug administration . Therefore, prolonged and acute administration of the p38 MAPK inhibitor blocked apparent spontaneous pain, but was not sufficient to block the tumor-induced tactile hypersensitivity. Notably, our findings are consistent with a recent clinical report in which systemic administration of a p38 MAPK inhibitor (dilmapimod) to humans with neuropathic pain diminished pain intensity compared to placebo, but failed to alter measures of evoked pain, including tactile allodynia and altered thermal thresholds .