Long non-coding RNA NKILA inhibits migration and invasion of non-small cell lung cancer via NF-κB/Snail pathway

Lung cancer is the most common incident cancer and the leading cause of cancer death all over the world [1]. More than 85% of those cases are currently classified as non-small-cell lung cancer (NSCLC). Although remarkable progress during the past decades in diagnose and treatment, including traditional surgery, chemotherapy, radio-therapy as well as emerging target therapy and immune therapy, the predicted 5-year survival rate is only around 15% - a figure that has only marginally improved during the past few decades [2, 3]. And it is mainly a consequence of regional recurrence and lymph node metastasis. Therefore, a thorough understanding of the molecular mechanism involved in the development and progression of NSCLC could provide more effective diagnostic markers and targets for NSCLC patient therapy.

It is now well known that only less than 2% of the genome encodes proteins, but at least 75% is actively transcribed into noncoding RNAs [4]. Although some of the noncoding transcripts are small, such as miRNA, piRNA and snoRNA, most of them larger than 200 nucleotides in length, which were catalogued as long noncoding RNAs (lncRNAs) [5]. LncRNAs are a highly heterogeneous group of transcripts that regulate gene expression via diverse mechanisms. Consequently, a large amount of lncRNA had been found being dysregulated in a range of caners and contributing to tumorigenesis and tumor progression [6]. For example, the Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), one of the most highly abundant lncRNAs, was upregulated in NSCLC and correlated with metastasis. The overexpression of MALAT1 can promote the metastasis of lung cancer cells by regulate downstream gene expression and alternative splicing [7]. In contrast, the long non-coding RNA PVT1 suppressed cell growth and induced apoptosis by binding to the enhancer of zeste homolog 2 (EZH2) protein, a histone methyltransferase of the PRC2 complex, in NSCLC [8]. So far, even the exact mechanism of lncRNAs still unknown, most of the lncRNAs exert bio-function via interact with other molecules [9] including mRNA, miRNA, genome DNA, other lncRNAs and mostly proteins, especially proteins in vital signal pathways.

NF-KappaB Interacting LncRNA (NKILA), encoded by a gene at chromosome 20q13 just near by the prostate transmembrane protein androgen induced 1 (PMEPA1) (also named solid tumor associated gene 1, STAG1) [10], is upregulated by NF-κB in breast cancer [11]. Previous study found NKILA can suppress the migration and invasion of breast cancer and tongue squamous cell carcinoma cells via directly binding with NF-κB: IκB complex, thereby inhibiting IKK-induced IκB phosphorylation and NF-κB activation [11, 12]. However, the clinicopathological correlation and functional role of NKILA in NSCLC is still elusive. Moreover, PMEPA1 expression is strongly enhanced by TGF-β and negative feedback inhibit TGF-β/Smad signal pathway in return [13‐15], which brings the hypothesis that NKILA may also participate in the TGF-β signal pathway. In this study, we aimed to investigate the role of NKILA in TGF-β pathway, regulating the NF-κB activity and tumor cell EMT in NSCLC, as well as the clinical significance of NKILA in predicting lymph node metastasis.

With the development of next-generation sequencing and applying to a growing number of cancer transcriptomes has indeed revealed thousands of lncRNAs whose aberrant expression is associated with different cancer types. Among the few that have been functionally characterized, a considerable number of lncRNAs have key roles in tumor signal pathway regulation and thus affect various aspects of tumorigenesis and progression, including proliferation, survival, migration or genomic stability [17]. Unlike mRNAs, lncRNAs are more often conserved of short sequence motifs or secondary structure and act as a molecular scaffolds that interact with multiple regulatory proteins [9]. For example, it was reported that NRON, as a molecular scaffold, is necessary for the assembly of NFAT and several NFAT kinases, which are responsible for NFAT phosphorylation and its sequestration in the cytosol [18]. In contrast, NKILA binding with NF-κB: IκB complex and masking the phosphorylation sites of IκB from IKK to inhibit the IKK-induced IκB phosphorylation [11], which was indirectly verified in NSCLC via RIP assay. Consistent with previous research, we further found overexpression of NKILA can inhibit the phosphorylation of IκB and NF-κB translocation to nucleus in NSCLC cells.

As a transcription factors, NF-kB regulates many downstream genes expression including that regulate cell metastasis and plays pivotal roles in both promoting and maintaining an invasive phenotype. In our study we found increasing NKILA expression in NSCLC cells can significantly inhibit cell migration invasion and proliferation. Silencing NKILA expression in A549 and H226 obviously increased the cell viability, migration and invasion ability. However, these phenotypes can be reversed by NF-kB inhibitor. These results suggested that NF-κB is responsible for NKILA-regulated malignant phenotype.

In mammalian cells, Snail has been shown to be a direct repressor of E-cadherin gene transcription though specific E-boxes in the proximal promoter [16] as well as upregulate the expression of N-cadherin and vimentin [19], which are the hollmarks of EMT. As a critical regulator of EMT signaling pathways, overexpression of Snail correlates with tumor grade, nodal metastasis, and tumor recurrence and predicts a poor outcome in patients with various cancers [20‐25]. The expression of Snail can be regulated by NF-κB through both transcriptional and post-translational mechanisms [26]. On the one hand, NF-κB can bind to the human Snail promoter and increase Snail transcription directly [27]. On the other hand, NF-κB can blocks the ubiquitination and degradation of Snail, which is required for inflammation-induced cell migration and invasion [28]. Knockdown of Snail expression inhibited cell migration and invasion induced by inflammation, which mainly owe to NF-κB [28]. In lung adenocarcinoma the TGF-β induced EMT can be inhibited by suppressing NF-κB mediated Snail activation [29]. All these finding can demonstrates that Snail plays fundamental role in NF-κB-regulated migration, invasion, and metastasis. In our study we also found when overexpressed NKILA, which attenuated the NF-κB activation, the Snail expression was decreased accompany with increase of E-cadherin level and decrease of N-cadherin and Vimentin, as well as weakened migration and invasion ability. Contrarily, knockdown of NKILA increased the expression of snail followed with attenuation of E-cadherin and enhanced N-cadherin and vimentin expression, which obviously increased the malignant phenotype of NSCLC cells. What’s more the influence on Snail, EMT markers and cell mobility caused by NKILA can be abolished by NF-κB inhibitor. Collectively, we can draw a conclusion that the NKILA regulated migration and invasion is mediated by NF-κB/Snail signal pathway.

TGF-β and NF-κB, two vital signal pathway in cancer, have complex interaction between each other. TGF-β can active NF-κB signal pathway via phosphorylating IKK2 and NIK [30], on the other hand NF-κB mediated Smad7 induction inhibits Smad2/3 activation [31]. LncRNA, as an emerged crucial regulators of various biological processes, present strong ability to link different signal pathway together, and supplement the signal network of cancer [32]. For example, the TGF-β1-upregulated lncRNA malat1 can active PI3K/AKT pathway and promote the proliferation and invasion of human osteosarcoma [33‐35]. For the genome location of NKILA just besides PMEPA1, which has been proven being upregulated by TGF-β [15, 36],and play an important role in NSCLC [37, 38]. In our study, we found NKILA can be dramatically upregulated by TGF-β1 treatment in NSCLC cells. However, contrary to earlier reports in breast cancer, just no more than 2-fold increase following TNF-α or IL1β treatment. What’s more, the TGF-β1 induced NKILA expression can’t be reversed by NF-κB nuclear translocation inhibitor JSH-23, while, TNF-α or IL1β induced NKILA upregulation can be absolutely abrogated by TGF-β receptor inhibitor SB505124 (Additional file 1: Figure S3). Combining with the ChIP assay result shown in Fig. 4g we could draw the conclusion that NKILA is directly upregulated by TGF-β classical pathway, however, the NKILA expression alter brought by NF-κB pathway may indirectly through TGF-β signaling in NSCLC cells. Subsequently, increased NKILA expression can inhibit the activation of NF-κB and then suppress the expression of Snail, which is a critical role upregulated by TGF-β signal pathway. Our study provides a new plausible interaction between TGF-β and NF-κB signal pathway.

TGF-β pathway plays dual anti- and pro-tumoral roles in a large panel of cancer types including NSCLC. In early-stage tumors, the TGF-β classical pathway have relative high activation and prevents incipient tumorigenesis [39]. While, with accumulation of genetic alterations of the core components and tumor-suppressive arm of TGF-β pathway lead it converse from tumor-suppressive to tumor-promoting activities [40, 41]. In our study, we found NKILA was upregulated by classical TGF-β pathway and had higher expression level in early stages (TNM stageIandII) than advanced stages (TNM stage III and IV) of NSCLC (p = 0.048, Table 1), which was consistent with the dual roles of TGF-β in cancer. Even exact mechanism is still unclear, NKILA brought further understanding of the contextual pleiotropy of TGF-β pathway in NSCLC.

Finally, our findings have potential clinical application value. In recent years, lncRNAs has been attracted more and more attention as potential therapeutic target for cancer. Arun and colleagues provided an exciting example of the potential of noncoding RNAs as therapeutic targets in the treatment of breast cancer [42]. Almost all of the lncRNAs exert function via special secondary structure and functional motif, which can be artificially synthesized. Therefore, engineered ncRNAs containing the particular functional domains have potential to be used for therapy. The expression of NKILA is negatively correlated with NSCLC lymph node metastasis suggest NKILA could be an effective marker and target for antimetastasis therapies.

In summary, NKILA is activated by TGF-β and inhibits NSCLC cell migration and invasion by inhibiting IκB phosphorylation and NF-κB activation then suppressing Snail regulated EMT (Fig. 7). Furthermore, NKILA expression is negatively associated with tumor metastasis in patients with NSCLC. These observations provide a potential therapeutic target for metastasis of NSCLC.