- Top of page
- MATERIALS AND METHODS
- Author contributions
- Supporting Information
Various forms of cancer are associated with debilitating pain. Approximately one-third of adults who are actively receiving treatment for cancer and two-thirds of those with advanced malignant disease experience pain. Various types of carcinomas and sarcomas metastasize to skeletal bones and cause spontaneous bone pain, hyperalgesia (exaggerated pain) and allodynia (pain in response to a normally innocuous stimulus), which is accompanied by bone degradation and remodelling of peripheral nerves (Mantyh, 2004; Wacnik et al, 2001). In a large number of clinical cases, cancer-associated pain, particularly the neuropathic component thereof, is resistant to conventional therapeutics or their application is severely limited owing to the widespread side effects (Mantyh, 2006; Portenoy, 1999). In order to develop novel, mechanism-based therapeutic strategies, it is imperative to delineate the cellular and molecular mechanisms underlying cancer-induced pain. Indeed, although tumour-induced pain shares features of inflammatory as well as neuropathic pain, it is clearly distinguished by distinct pathophysiological and mechanistic aspects (Mantyh, 2004; Wacnik et al, 2001).
Non-coding RNAs (ncRNAs), including the more well-studied microRNAs (miRNAs), are emerging as critical modulators of normal cellular functions as well as pathological processes (Huttenhofer & Schattner, 2006; Huttenhofer et al, 2005; Mattick, 2004). Various diseases are associated with unique miRNA expression signatures, which can not only be exploited as diagnostic and prognostic markers, but also reveal deep insights into disease pathology. Moreover, miRNAs can act as ‘master switches’ of the genome to regulate the expression of diverse proteins and orchestrate multiple cellular pathways, thereby harbouring tremendous therapeutic potential.
Recently, neuropathic pain conditions have been suggested to deregulate the expression of miRNAs in pain pathways in profiling studies (Aldrich et al, 2009; Bai et al, 2007; Imai et al, 2011; Kusuda et al, 2011; Poh et al, 2011; von Schack et al, 2011). Moreover, specific miRNAs have been associated with inflammatory pain and the deregulation of ion channel expression in sensory neurons in rodent models of inflammatory and neuropathic pain (Favereaux et al, 2011; Li et al, 2011; Zhao et al, 2010). However, to date, nothing has been reported on the modulation of miRNA expression in conjunction with cancer pain. Moreover, previous studies on miRNA deregulation in pain have mostly remained at the niveau of profiling expression; in contrast, the functional consequences of miRNA deregulation, their downstream targets and the mechanisms by which miRNAs regulate processes modulating nociception have not been resolved.
Both peripheral as well as spinal contributions are of critical importance in understanding cancer pain and developing therapeutic approaches (Gordon-Williams & Dickenson, 2007; Mantyh, 2004). Our key interest is to address plasticity mechanisms at the interface between tumour cells and nociceptive pathway; thereby, mechanisms operational in sensory neurons are of prime interest. This is supported by numerous studies, which have demonstrated changes in the structure as well as the function of sensory neurons in cancer pain states, which are attributed to effects of tumour growth and tumour-associated mediators (Cain et al, 2001; Constantin et al, 2008; Mantyh, 2004).
Starting with a genome-wide screen for miRNAs regulated in sensory neurons of the dorsal root ganglia (DRG) in the state of bone-metastatic pain, we functionally validated a set of prominently regulated miRNAs and report the impact of deregulating their expression in sensory neurons on cancer-associated nociceptive hypersensitivity. Our in silico analysis of gene targets of prominently regulated miRNAs not only revealed that several prominent genes encoding known nociceptive mediators, but also uncovered a novel target encoding a chloride channel, which we functionally validated as an important modulator of nociceptive sensitivity. Our results underscore the importance of miRNA regulation in sensory neurons in the context of bone metastatic pain and systematically delineate the potential of ncRNAs as druggable targets for future treatment of cancer-associated pain.
- Top of page
- MATERIALS AND METHODS
- Author contributions
- Supporting Information
Despite recent advances, cancer pain remains a major challenge for clinicians and basic scientists and there is an urgent demand for the development of specific mechanism-based therapies. Using a comprehensive approach combining genome-wide miRNA screening, molecular and in silico analyses with behavioural approaches in a clinically relevant model of metastatic bone-cancer pain, we now show that tumour-induced hypersensitivity is associated with a dysregulation of miRNA expression in sensory neurons corresponding to tumour-affected areas. This is the first study profiling genome-wide miRNA expression and their functional importance in the development and maintenance of tumour-mediated chronic pain. Furthermore, whereas previous studies on the involvement of miRNAs in other forms of pain disorders, e.g. neuropathic pain, have either addressed miRNA profiling without functional analysis or have focussed on a single target gene modulated by a miRNA (Aldrich et al, 2009; Bai et al, 2007; Favereaux et al, 2011), the present study spans multiple levels of analysis starting with a genome-wide identification of the miRNA regulatory network of cancer pain-associated miRNAs in sensory neurons, their functional validation in vivo all the way to the identification, molecular characterization and functional validation of a novel target gene.
Our in vivo analyses revealed that the expression of 57 miRNAs amongst the 615 tested mouse miRNAs is dysregulated in sensory neurons of the DRG directly under the influence of tumour growth in their innervation territory, representing a novel and intriguing aspect of tumour–nerve interactions. Interestingly, miRNA dysregulation only became evident when pronounced tumour-induced hypersensitivity was established, but did not manifest at the beginning stages (e.g. 4 days post-implantation). This suggests that early periods of tumour pain likely result from local interactions between tumour cells and nerves, which lead to sensitization of nerve afferents, and that miRNA dysregulation and the resulting changes in the expression of a multitude of genes in sensory neurons rather contribute to the maintenance and long-term nature of tumour pain. This study was based upon a model involving tumour-induced remodelling of bone and ensuing mechanical hypersensitivity in the adjacent skin. It will be interesting in future studies to compare miRNA expression profiles with models based upon direct injection of tumour cells in the skin to work out the component of bone pain.
In contrast to tissues such as liver, heart, blood vessel and the lymphatic system, amongst others, which are amenable to modulation via exogenously delivered miRNA modulators (Bernardo et al, 2012; Krutzfeldt et al, 2005, 2007; Muramatsu et al, 2013; Pullamsetti et al, 2012; Yigit et al, 2012), the nervous system is notoriously difficult to target (Long & Lahiri, 2012). To disrupt the pathophysiological induction or downregulation of miRNA in DRGs in vivo, we established effective protocols for intrathecal delivery of miRNA inhibitors or mimics and demonstrated the efficacy of selective manipulations in miRNA expression in vivo. Our behavioural analysis in the bone-metastases model indicated that inhibiting the tumour-induced upregulation of miR-1a-3p or miR-34c-5p, but not of miRNA-544-3p, in sensory neurons markedly attenuated tumour-mediated hyperalgesia. Furthermore, reversing pathophysiological decrease of miR-483-3p, but not of miR-291b-5p, attenuated tumour-mediated hyperalgesia. In contrast, augmenting the expression of miR-370-3p in DRGs led to exaggerated tumour-mediated hyperalgesia. Thus, the approach established and described in this study provides proof-of-principal that it is effective and valuable in elucidating the functional contributions of miRNAs. An intriguing observation is that even though injection of miRNA modulators was stopped at PID-9, the effect persisted until PID-15. As it has been shown that short-oligonucleotides can be detected in the DRGs up to 24 h following injection (Layzer et al, 2004; Mook et al, 2010), the change in miRNA expression will persist until at least 24 h thereafter, i.e. PID-10. Since there is a time lag between miRNA action and a change in the expression of the protein product of the final target gene(s), the physiological effect resulting from altered protein function could come about over several days thereafter. Another interesting finding from our profiling analyses is, among 43 potentially novel, unannotated miRNAs (classified as solexa sequences) several were observed to be strongly expressed in DRG and 10 showed significant dysregulation in expression in tumour-bearing as compared to sham-treated mice. It will be interesting in future studies to systematically elucidate the contributions of all annotated as well as novel miRNAs which were found to be dysregulated in conditions of bone metastatic pain.
Here, we chose miRNA-1a-3p as our prototypic miRNA for detailed mechanistic analyses owing to several reasons. One, although miR-1a-3p was characterized to be a non-neuronal miRNA with expression levels in the central nervous system 100- to 1000-fold lesser when compared to cardiac tissue (Mishima et al, 2007), later studies showed that miR-1a-3p is expressed in sensory neurons of the DRG in mice as well as humans (Bastian et al, 2011), which imparts it a particular translational significance; moreover, it has been well-characterized via in situ hybridization analysis to be localized to peptidergic nociceptors (Bastian et al, 2011). Secondly, mimicking miR-1a-3p in neuronal cultures attenuates neurite outgrowth (Bastian et al, 2011). Third, our results on regulation of miR-1a-3p expression in bone metastatic pain emerged to be intriguingly different to known findings in the context of neuropathic and inflammatory pain; whereas inflammatory pain as well as partial sciatic ligation are associated with a decrease in miR-1a-3p expression in the DRG and spinal cord, sciatic nerve axotomy induces a robust increase in miR-1a-3p expression in the DRG and a corresponding decrease in the spinal cord (Kusuda et al, 2011). Here, we observed that miRNA-1a-3p was highly upregulated in the DRG in bone metastatic pain. Despite insights into it's dysregulation in inflammatory and neuropathic states, the functions of miR-1a-3p in pain modulation had not been analysed so far. Our in vivo analyses revealed as miR-1a-3p in the DRG to be a functionally important positive modulator of bone metastatic pain, indicating a pronociceptive function for miR-1a-3p.
Based on these intriguing findings, we sought to identify genes regulated by miR-1a-3p in DRG neurons in an effort to work out mechanistic details. In doing so, we established and validated an approach to narrow down and select relevant genes from the very long lists of target genes, which typically emerge from in silico analyses. Thus, out of 62 mRNAs putative targets for miR-1a-3p predicted by at least 2 out of 14 algorithms applied, only 25 showed at least 50% up-regulation following miR-1a-3p inhibition in sensory neurons in vivo, thereby cautioning about the strength of interpretations that can be made from in silico analyses alone. Interestingly, except for Hand2 and Igf1, almost all of genes that were functionally validated as miR-1a-3p targets in other systems were not found to be regulated by miR-1a-3p inhibition in sensory neurons, thereby highlighting the context-dependence of functionality of miRNA function and gene regulation. One of the most interesting aspects of this study was the identification of Clcn3, a chloride channel, as a functionally important gene target of miR-1a-3p in sensory neurons of the DRG. Apart from evidence for miRNA-1a-3p binding to the 3′UTR of Clcn3 and regulation of it's translation, the finding that the expression of Clcn3 expression is reciprocally regulated with respect to miR-1a-3p expression in the DRG following peripheral tumour induction established Clcn3 as a miR-1a-3p target in sensory neurons. Furthermore, the phenotype of exaggerated tumour-mediated hyperalgesia evoked by specifically knocking Clcn3 expression down in sensory neurons in vivo matched perfectly with attenuation of tumour-mediated hyperalgesia evoked by miR-1a-3p knockdown in the DRG. There are several ways via which Clcn3-mediated Cl− flux could affect sensory neuron function. For example, it has been shown to be required for lysophosphatidic acid (LPA)-activated Cl− current activity in myofibroblast differentiation (Yin et al, 2008), a molecular pathway which may be relevant to sensory neurons given that LPA is a highly potent and important modulator of sensory neuron function in pain disorders (Inoue et al, 2004; Ueda, 2008). Importantly, Clcn3 plays a role in both acidification and transmitter loading of GABAergic synaptic vesicles, which is very interesting in the light of the important role for GABAergic function in sensory terminals of nociceptors (Witschi et al, 2011). Moreover, in brain tissue, functional interactions between Clcn3 and Ca2+/calmodulin-dependent protein kinase II have been reported (CamKII) (Cuddapah & Sontheimer, 2010; Huang et al, 2001), which could be relevant to the pain modulatory role of CamKII (Crown et al, 2012). Thus, the miR-1a-3p-Clcn3 miRNA–mRNA regulation pair holds tremendous potential for modulating pain over multiple ways. A more detailed analysis of Clcn3 expression and sub-cellular distribution in the future could be very insightful; similarly, it will be interesting to study whether the role of Clcn3 observed here in the context of cancer pain also extends to the modulation of inflammatory or neuropathic pain states, given especially our observation that knocking down the basal expression of Clcn3 by itself significantly impacted on mechanical sensitivity in the absence of tumour growth.
Here, miR-34c-5p emerged as another key pronociceptive miRNA, which is induced in sensory neurons of the DRG in bone metastatic pain. Induction of this miRNA has been previously implicated in the hippocampus in the context of memory impairment and in the amygdala in the context of stress (Haramati et al, 2011; Zovoilis et al, 2011). Several notable pain modulatory genes are to be found amongst the in silico prediction list of miR-34c-5p target genes, including Cacnb3 (the gene encoding calcium channel, voltage-dependent, beta 3 subunit), Gabra3 and Gabra1 (encoding GABA-A receptor, subunits alpha 3 and 1, respectively), Scn2b (sodium channel, voltage-gated, type II, beta), Bdnf (encoding brain derived neurotrophic factor), Calca (encoding calcitonin/calcitonin-related polypeptide, alpha), Il6st (encoding interleukin 6 signal transducer), Ednrb (endothelin receptor type B), amongst many others, which are known to significantly impact on pain (De Jongh et al, 2003; Enna & McCarson, 2006; Griswold et al, 1999; Knabl et al, 2008; Li et al, 2012; Murakami et al, 2007; Obata & Noguchi, 2006; Pedersen et al, 2007; Quarta et al, 2011; Waxman, 2010). Similarly, miR-370-3p, which was known in the context of angina pectoris and tumour suppression so far (Hoekstra et al, 2010; Zhang et al, 2012), emerged as a pro-nociceptive miRNA. Our in silico analyses revealed many interesting nociception related genes have binding sites for miR-370-3p, including gene encoding the purinergic receptor P2X, ligand-gated ion channel, 3 (P2rx3), opioid receptor, delta 1 (Oprd1), runt related transcription factor 1 (runx1), calcium channel, voltage-dependent, alpha 2/delta subunit 2 (Cacna2d2) among others, which have been characterized functionally in previous studies (Abrahamsen et al, 2008; Boroujerdi et al, 2011; Chen et al, 2006; Scherrer et al, 2010; Taylor & Garrido, 2008). Further detailed analyses will reveal which of these putative targets mediate the pronociceptive functions of miR-34c-5p and miR-370-3p in the context of cancer pain. Finally, two of the six dysregulated miRNAs analysed did not reveal a functional impact on tumour hypersensitivity. This is, however, not surprising since tumour–nerve interactions can encompass many other functional changes, e.g. cell death in the DRG, neuropathy, immune infiltration, etc., which were not studied here as readouts and represent interesting options for further detailed analyses.
Thus, the results of this study deliver valuable new insights into mechanisms of cancer pain. Furthermore, they open up very attractive therapeutic options since potential caveats associated with miRNAs and miRNA derivatives, e.g. ‘off-target’ effects, can be overwhelmed in this fast-developing field on the way towards the therapeutic development. Importantly, ncRNA-based diagnostics and therapeutics may have superior advantages by targeting multiple pain-associated genes simultaneously, and this study provides a preclinical basis in the context of cancer-associated pain.