Accumulating evidence has uncovered roles for LncRNAs in the clinico-pathological features of various cancers, including potential roles for dysregulated LncRNAs as biomarkers for diagnosis, prognosis and therapeutic targeting(23, 24). We previously revealed that LncRNAs are dysregulated in human GC(11); however, the roles of LncRNAs in GC remained to be elucidated. In this study, we defined a novel LncRNA, DRD5P2, which functions as a tumor suppressor in GC. We presented data showing that DRD5P2 is significantly downregulated in GC tissues and that reduced expression of DRD5P2 is correlated with advanced GC and poor prognosis, indicating that DRD5P2 plays an important role in GC progression and could potentially serve as a prognostic indicator for GC. Furthermore, we provided evidence for a pivotal role of DRD5P2 in EMT, a process that facilitates cancer metastasis by conferring enhanced migrative and invasive ability to tumor cells during tumor progression (25, 26). Snail functions as a key transcription factor in the EMT process by repressing E-cadherin expression, and epigenetic modulation of the Snail-mediated EMT program by LncRNAs has been studied previously(27). Consistently, in this study, we found that ectopic expression of DRD5P2 in GC cells leads to suppression of Snail transcription and EMT, indicating that DRD5P2 functions mechanistically in GC as a powerful suppressor of EMT.
Signaling pathways that regulate the EMT program are highly complicated and can be activated by various dynamic stimuli from the local microenvironment, including growth factors and cytokines (TGF-β, Wnt, FGF, et al), hypoxia, and the extracellular matrix (ECM). Previous studies have implicated ERK signaling in the activation of EMT, and inactivation of ERK has been shown to impair the suppression of E-cadherin expression(28, 29). In this study, we revealed that abrogating the activation of ERK with U0126 leads to the inhibition of EMT in DRD5P2-deficient GC cells, which suggests that the inhibition of EMT by DRD5P2 is mediated by inactivation of the ERK signaling pathway. ERK can phosphorylate CREB at ser133 to activate its transcriptional activity(30). We demonstrated that upon ERK pathway activation in DRD5P2-deficient GC cells, CREB binds to the Snail promoter and promotes Snail transcription. Thus, our data highlight a necessary role for ERK/CREB signaling pathway in the regulation of EMT by DRD5P2.
LncRNAs mainly exert their functions upon the formation of LncRNA-protein complexes. In this study, we determine that DRD5P2 binds to ROCK2, a member of a family of serine-threonine kinases. ROCK2 has been described as a critical mediator in regulating cancer cell mortality and metastases, and its overexpression is correlated with poor clinical outcome and chemo-resistance in various cancers (15, 31). Here, integrative analysis including RNA-pulldown-MS and RIP assays in GC cells identified ROCK2 and the E3 ligase KAP1 as binding partners of DRD5P2 in GC cells. Further co-IP experiments revealed that DRD5P2 overexpression promotes ROCK2 ubiquitin by recruiting KAP1. Activation of ERM (Ezrin-Radixin-Moesin) proteins has been characterized as a downstream event of ROCK2 expression(32), leading to RAS activation by Ezrin(33). In our study, we found that ROCK2 binds and activates Ezrin, which subsequently activates HRAS and ERK. Thus, our data uncover a comprehensive pathway leading to EMT regulation by a LncRNA in GC. Though our data are consistent with known mechanisms of EMT regulation, there are some limitations of our current study, including a limited sample size for validating the significance of the LncRNA DRD5P2/ROCK2/ERK/Snail axis in GC metastasis. Therefore, the application of our findings will need to be verified with a larger and more diverse cohort.
In this study, we also explored upstream mechanisms regulation DRD5P2 expression in GC, including hypoxia, which is a common feature of the cancer microenvironment. Cancer cells obtain enhanced abilities of migration, invasion, and glycolysis in hypoxic microenvironments. HIF-1α is one of the most well-known factors activated in cancer and regulates cancer progression by facilitating the expression of its target genes or non-coding RNAs (34–36). However, prior to this study, the role of HIF-1α in regulating LncRNA expression in GC remained to be elucidated. Here, we identified DRD5P2 as a hypoxia-related LncRNA and determined that DRD5P2 transcription is repressed in a HIF-1α/ZNF263-dependent manner under hypoxic condition. Recently, the HIF-1α RNA antagonist RO7070179 has been subjected to a phase I clinical trial (ClinicalTrials.gov, NCT02564614). Our study indicates that targeting the HIF-1α/ZNF263/DRD5P2 axis could be further developed as a strategy in GC treatment.
Collectively, the findings of the current study support a model in which LncRNA DRD5P2 binds to ROCK2 and mediates its degradation by recruiting KAP1 and then inactivates the ERK/CREB pathway by inhibiting Ezrin/HRAS signaling, which in turn leads to reduced Snail transcription and inhibition of EMT.