LncRNA DUXAP9‐206 directly binds with Cbl‐b to augment EGFR signaling and promotes non‐small cell lung cancer progression

Abstract Long noncoding RNAs (lncRNAs) are involved in the pathology of various tumours, including non‐small cell lung cancer (NSCLC). However, the underlying molecular mechanisms of their specific association with NSCLC have not been fully elucidated. Here, we report that a cytoplasmic lncRNA, DUXAP9‐206 is overexpressed in NSCLC cells and closely related to NSCLC clinical features and poor patient survival. We reveal that DUXAP9‐206 induced NSCLC cell proliferation and metastasis by directly interacting with Cbl‐b, an E3 ubiquitin ligase, and reducing the degradation of epidermal growth factor receptor (EGFR) and thereby augmenting EGFR signaling in NSCLC. Notably, correlations between DUXAP9‐206 and activated EGFR signaling were also validated in NSCLC patient specimens. Collectively, our findings reveal the novel molecular mechanisms of DUXAP9‐206 in mediating the progression of NSCLC and DUXAP9‐206 may serve as a potential target for NSCLC therapy.

remains poor. 3 Thus, exploration of the molecular mechanisms and molecules involved in the development and progression of NSCLC may provide valuable therapeutic targets for these diseases.
It has been shown that protein-coding genes occupy only a small proportion (1%-2%) of the human genome. The rest are nonprotein-coding transcripts including small noncoding RNAs and long noncoding RNAs (lncRNAs). 4 LncRNAs have gradually been shown to be pivotal molecules that affect cancer development and progression. 5 The length of lncRNAs is longer than 200 nucleotides (nt), yet this class of RNA has limited coding potential. 6 LncRNAs function in a wide range of biological processes and can regulate gene expression through diverse mechanisms. 7 It has been suggested that cytoplasmic lncRNAs are important molecules in regulating cancer intracellular signaling pathways. 8 For example, the lncRNA MAYA modulates the methylation status of MST1 leading to bone metastasis. 9 The lncRNA lncAKHE enhances cell growth and migration in hepatocellular carcinoma via activation of NOTCH2 signaling. 10 Epidermal growth factor receptor (EGFR), a transmembrane glycoprotein with tyrosine kinase activity is often aberrantly activated by mutation or overexpression in many human cancers. 11 Over 60% of the NSCLC cases show EGFR overexpression, which is associated with poor prognosis of NSCLC. 12,13 Ligand-activated EGFR leads to stimulation of intracellular cascades, such as the RAS/RAF/ERK and PI3K/AKT signaling pathways. Abnormally activated EGFR drives the malignant phenotype including cell proliferation, survival, invasion and migration. 13 Although several lncRNAs have been reported to modulate tumour proliferation, apoptosis or metastasis, the specific mechanisms of lncRNA involvement in EGFR signaling pathway-

| Tissue specimens
A total of 216 paraffin-embedded NSCLC specimens, were clinically and histopathologically diagnosed at the Sun Yat-sen University Cancer Center. These specimens are defined as the SYSUCC cohort.
NSCLC tissue and paired adjacent non-cancerous lung tissue specimens were stored frozen in liquid nitrogen until further use. Adjacent non-tumour tissue specimens were taken a standard distance (3 cm) from the margin of resected tissues of patients with NSCLC who underwent surgical lung resection and confirmed by pathological diagnosis. For the use of these clinical materials for research purposes, prior patient consents and approval from the Institutional Research Ethics Committee of the Sun Yat-sen University were obtained. Clinical information of the samples is described in detail in Table S1.

| Small interfering RNAs
All siRNAs were synthesized by RiboBio (Guangzhou, China) and used at a final concentration of 100 nmol/L. The sequences of the siRNAs are summarized in Table S3.

| 5′ and 3′ rapid amplification of cDNA ends PCR
Total RNA extracted from human A549 and H1703 cells was subjected rapid amplification of cDNA ends polymerase chain reaction (RACE PCR) (Ambion, Austin, TX, USA) according to the manufacturer's specifications. Gene-specific primers used for the PCR of RACE analysis are listed in Table S4.

| Cell nucleus/cytoplasm fraction isolation
Cell nucleus/cytoplasm fraction isolation was performed using Nuclear and Cytoplasmic Extraction Kit (Ambion). In brief, cells were washed with ice-cold PBS two times and then ice-cold CERI, CERII and NER reagents were added sequentially. After vortex and short centrifugation, the supernatant was collected as cytoplasmic fraction and the remainder with additional washing was considered as nuclear pellets.   Table S5.

| Co-immunoprecipitation
The indicated cells were collected and lysed with cell lysis buffer

| RNA pull-down assay
A T7 High Yield RNA Synthesis Kit (Ambion) was used to obtain in vitro transcribed RNA according to the manufacturer's instructions. The transcribed RNA was labelled using a 3′ End Biotinylation Kit (Thermo) and the protein that interacted with the RNA was enriched with an RNA-Protein Pull-Down kit (Thermo) according to the manufacturer's instructions. In brief, biotinylated labelled RNA was incubated with washed streptavidin beads at 4°C. Then, cell lysate was added to the binding reaction and further incubated overnight. Retrieved proteins were detected by Western blotting analysis or mass spectrometry (MS) identification.

| Wound healing and transwell assays
For wound healing assays, the indicated cells were plated in 6-well plates and then, streaks were created with a pipette tip. After

| Cell proliferation assay
For colony formation assays, the indicated cells were plated in sixwell plates (5 × 10 2 cells) and cultured for 10 days. Then, the cells were fixed with 4% formaldehyde for 15 minutes and stained with 0.1% crystal violet for 10 minutes.
For 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, a total of 1 × 10 3 cells were seeded in 96-well plates, stained with 100 mL of sterile MTT dye (0.5 mg/mL, Sigma) for 4 hours at 37°C, followed by removal of the culture medium with addition of 150 mL of dimethyl sulfoxide (Sigma). The samples were shaken at room temperature for 10 minutes and then, the absorbance of the stained cells was measured at 570 nm, with 655 nm as the reference wavelength.

| In situ hybridization and immunohistochemistry
Formalin-fixed, paraffin-embedded NSCLC samples were fixed in 4% paraformaldehyde and incubated with proteinase K for 20 minutes at 37°C. After they were dewaxed and rehydrated, the samples were hybridized with 40 nmol/L digoxin-labelled DUXAP9-206 probe (Exiqon, for sequence, see Table S6) at 55°C overnight. The slides were then washed with SSC buffer and incubated with anti-digoxin monoclonal antibody (Roche) for 1 hour at room temperature followed by staining with Nuclear Fast Red solution.
Immunohistochemistry assays were performed and quantified as previously described 17  The staining index (SI) was calculated as the product of staining intensity × proportion of positively stained cells resulting in scores of 0, 1, 2, 3, 4, 6 or 9. An SI score of 3 was used as a cut-off value based on heterogeneity measurement using the log-rank test with respect to overall survival for the expression of DUXAP9-206 or the protein of interest.

| Statistical analysis
All statistical analyses were carried out using the SPSS 20.0 statistical software package. Survival curves were analysed by the Kaplan-Meier method and a log-rank test was used to assess significance.
The correlation between the expression levels of DUXAP9-206 and clinical parameters of patients was assayed by a chi-squared test.
Student's t test was used to compare between groups. In all cases, error bars represent the mean ± SD derived from three independent experiments. P values <0.05 were considered statistically significant.

| DUXAP9-206 is up-regulated in NSCLC and correlates with patients' survival
To explore the functional and clinical relevance of DUXAP9-206 in NSCLC, we initially analysed the expression levels of DUXAP9-206.
In situ hybridization (ISH) analysis using a specific probe revealed that the expression levels of DUXAP9-206 were significantly elevated in NSCLC tumour tissues compared with paired adjacent nontumour tissues ( Figure 1A). The level of DUXAP9-206 in NSCLC tissues was further verified by qRT-PCR assays with specific primer ( Figure 1B). Consistently, DUXAP9-206 was highly expressed in NSCLC cell lines compared with normal lung epithelial cells (BEAS-2B) ( Figure 1C). Moreover, in a set of 216 NSCLC patients in the Sun Yat-sen University Cancer Center (SYSUCC) cohort (Table S1) for whom overall survival data were available, patients with high  Figure 1G).
Using RT-PCR of nuclear and cytoplasmic fractions, we found that DUXAP9-206 was mainly located in the cytoplasm of NSCLC cells ( Figure 1H).   Taken together, these data strongly suggest that DUXAP9-206 may act as a driver molecule to promote NSCLC metastasis and proliferation.

| DUXAP9-206 directly interacts with Cbl-b in the cytoplasm
We next sought to explore the possible mechanism underlying the role of DUXAP9-206 in regulating NSCLC progression in cytoplasmic processes. Previous studies have extensively shown that many

| DUXAP9-206 binds with Cbl-b to prevent degradation of EGFR
Cbl-b, a member of the Cbl protein family, functions as an E3 ubiquitin-protein ligase, which binds with activated tyrosine kinases, such as the EGFR, for lysosomal degradation leading to cellular responses including cell proliferation, motility/migration and invasion. 18,19 Combining the above results, it implicates a possibility that DUXAP9-206 may promote NSCLC progression by directly binding with Cbl-b to reduce the degradation of EGFR. To test this, we performed Co-IP assays in cells with stable overexpression or knockdown of DUXAP9-206 pretreated with the lysosome inhibitor chloroquine followed by EGF (100 ng/mL) treatment for 30 minutes. As shown in It has been well documented that ubiquitination and lysosomal degradation of EGFR requires direct binding with Cbl-b. EGFR is activated by binding with the ligands EGF, TGFα or HB-EGF and then, the EGFR-ligand complexes may be internalized from the plasma membrane to the endosomes and ubiquitinated EGFR is efficiently targeted to lysosomes for degradation. 20,21 The recruitment of an E3 ubiquitin ligase, Cbl-b, to activated EGFR is a key link that promotes its ubiquitination. 22,23 Cbl-b, a member of the Cbl proteins, which include c-Cbl, Cbl-b and Cbl-3 functions as a ubiquitin protein ligase (E3) for activated tyrosine kinases, such as EGFR and targets them for degradation. 24,25 Cbl-b-mediated EGFR degradation requires direct binding to the activated EGFR. [26][27][28] Finally, EGFR signaling-regulated biological effects, such as tumourigenesis and metastasis are attenuated via ubiquitination upon Cbl-b. 29 30 Jiang et al demonstrated that the lncRNA lnc-EGFR specifically bound to EGFR and blocked its ubiquitination by c-Cbl, a member of the Cbl family. 31 In the present study, we found that the expression of Cbl-b did not change when DUXAP9-206 was overexpressed, but the interaction of DUXAP9-206 and Cbl-b was increased leading to a decrease in the binding of Cbl-b to EGFR and consequently reducing the degradation of EGFR. Of note, this is the first report to identify an lncRNA that is directly involved in regulating the association of Cbl-b and EGFR, further enriching and improving the degradation mechanism of EGFR. According to previous reports, Cbl proteins contain several highly conserved domains including an N-terminal tyrosine kinase binding (TKB) domain and a RING finger. Cbl-b-mediated EGFR degradation requires the binding of the TKB domain to the activated EGFR. 29 It is of interest to further explore whether DUXAP9-206 also binds with the TKB domain of Cbl-b, thus preventing the interaction of the TKB domain of Cbl-b with EGFR. In addition, previous studies have reported that Cbl-b can regulate a cohort of target genes involved in different signaling pathways. 32,33 Whether the oncogenic role of DUXAP9-206 also requires additional mechanisms remains to be clarified in future studies. Furthermore, the clinical relevance of Cbl-b inhibitors currently in development suggests that DUXAP9-206 may indeed be a biomarker for response in NSCLC. It is of interest to further explore the role of these inhibitors in our presented models to determine if this is true. Taken together, our findings may further expand our understanding of the degradation process of EGFR in NSCLC and lay the foundation for identification of new therapeutic targets in the future.
Majority of the NSCLC patients worldwide are EGFR wild type. 34 However, numerous receptor tyrosine kinase inhibitors target mutation-positive NSCLC 35,36 and wild type EGFR patients lack effective treatments that significantly improve their survival and prognosis. 13,34 In this study, DUXAP9-206 was found to inhibit the degradation of EGFR in EGFR wild type NSCLC cells, such as A549 and H1703, indicating that DUXAP9-206 may be a promising therapeutic target for EGFR wild type patients in NSCLC. To further validate this result, we will collect wild type EGFR specimens from patients with NSCLC to compare the expression of DUXAP9-206 with normal patient specimens, verify their relationship with relevant signaling pathways and analyse the correlation with survival and prognosis of patients. Whether this mechanism also plays an important regulatory role in the EGFR mutant patients should be investigated further.
In summary, our study indicates that DUXAP9-206 and its regulated pathway is crucial for NSCLC and targeting DUXAP9-206 may be pivotal in the diagnosis or treatment of NSCLC.
F I G U R E 6 Clinical relevance of DUXAP9-206 with EGFR, p-EGFR, p-AKT and p-ERK in clinical specimens. (A) ISH analysis of DUXAP9-206 and immunohistochemical analysis of p-EGFR, EGFR, p-AKT and p-ERK expression in NSCLC tumour specimens. Sections were H&E stained to visualize the tumour structure and boundaries. Original magnification, ×400. (B) DUXAP9-206 expression was positively associated with expression levels of p-EGFR, EGFR, p-AKT and p-ERK in NSCLC specimens F I G U R E 7 Proposed functional action of DUXAP9-206 in modulating NSCLC proliferation and metastasis. LncRNA DUXAP9-206 directly binds with Cbl-b to augment EGFR signaling and promotes non-small cell lung cancer progression