CASC15 is highly expressed in NSCLC and is essential for tumor cell migration and growth
To uncover novel lncRNAs that are involved in the development of lung cancer, we reanalyzed a publicly available dataset involving sixty pairs of human lung carcinomas and adjacent normal lung tissues (GEO dataset GDS3837). Results showed that, CASC15, one lncRNA that was firstly identified as a tumor suppressor in neuroblastoma but was later reported to play an oncogenic role in melanoma[21, 22], was significantly upregulated in NSCLC compared with matched adjacent normal tissues (p < 0.001, Fig. 1a). Kaplan-Meier analysis of another dataset involving 293 lung tumor samples (GEO dataset GSE30219) revealed that high expression levels of CASC15 significantly correlated with a reduction in overall survival in NSCLC patients (log-rank test, p = 0.00054, Fig. 1b), suggesting that CASC15 upregulation might be crucial in NSCLC tumorigenesis and progression. To investigate whether the expression level of CASC15 is correlated with the malignant phenotype of NSCLC cells in vitro, we cultured five NSCLC cell lines and one normal human bronchial epithelial cell line BEAS-2B. Subsequent quantitative PCR (qPCR) analysis showed that CASC15 were widely overexpressed in NSCLC cell lines (Fig. 1c). Transwell migration assay revealed that siRNA-mediated knockdown of CASC15 resulted in a remarkable suppression of motility of A549 and H1299 cells, reducing the number of migrating cells to about 30% of that in the control group (Fig. 1d and Fig. S1). FITC-phalloidin staining followed by confocal microscopic imaging showed that CASC15 silencing caused an obvious reorganization of F-actin and morphological change from spindle to epithelioid in these two cell lines (Fig. 1e). Moreover, we performed CCK-8 assay and found that CASC15 knockdown led to a dramatic reduction in cell viability and proliferation of A549 and H1299 cells (Fig. 1f). To examine the effect of CASC15 on lung tumor growth in vivo, we established a subcutaneous xenograft model in nude mice using luciferase-expressing A549 and H1299 cells. One week after tumor cell inoculation, tumor growth were monitored by regular measurements using a digital caliper. As expected, stable knockdown of CASC15 significantly inhibited tumor growth in nude mice bearing human lung carcinoma xenografts (Fig. 1g). Taken together, these results supported the notion that CASC15 functions as an oncogenic lncRNA in NSCLC.
CASC15 exerts tumor-promoting effects in NSCLC cells mainly by upregulating its neighboring oncogene SOX4
The CASC15 intergenic locus, formerly known as the FLJ22536 or LINC00340 locus, spans over 500 kb between the SOX4 and PRL genes on chromosome 6p22.3 (Fig. 2a). To investigate whether CASC15 exerts tumor-promoting effects in NSCLC cells by influencing the expression of neighbouring genes[23], we firstly performed qPCR to analyze the expression levels of SOX4 and PRL upon CASC15 silencing. Results showed that knockdown of CASC15 in NSCLC cells markedly decreased SOX4 mRNA levels but had no effect on PRL expression (Fig. 2b). Further Western blotting analysis confirmed that CASC15 depletion caused significant downregulation of SOX4 at the protein level in both A549 and H1299 cells (Fig. 2c,d). Then, we performed CCK-8 assay and transwell migration assay to test the role of SOX4 in proliferation and migration of NSCLC cells. Knockdown of SOX4 also resulted in a remarkable suppression of proliferation and motility of A549 and H1299 cells (Fig. 2e,f and Fig. S2), to an extent similar to that caused by CASC15 knockdown (Fig. 1d,f). Moreover, ectopic expression of SOX4 largely abrogated the inhibitory effects on cell proliferation and migration mediated by CASC15 knockdown (Fig. 2g,h). In vivo experiments demonstrated that, compared with control groups with stable knockdown of CASC15, concurrent overexpression of SOX4 significantly promoted tumor growth in nude mice bearing human lung carcinoma xenografts (Fig. 2i and Fig. S3). To sum up, these results suggested that CASC15 exerts tumor-promoting effects in NSCLC cells mainly by upregulating its neighboring oncogene SOX4.
CASC15 activates Wnt signaling in NSCLC via SOX4-mediated stabilization of β-catenin protein
Previous studies have identified Wnt-β/catenin pathway as a regulatory target of SOX4 and that SOX4 functions to stabilize β-catenin protein[24, 25]. To determine whether CASC15 could activate Wnt signaling in NSCLC, we measured Wnt signaling activity by using the TOP-FLASH reporter assay in A549 cells, which was reported to express high levels of endogenous β-catenin[26]. Results revealed that β-catenin activity was dramatically reduced by CASC15 knockdown and was rescued by simultaneous overexpression of SOX4 in the same cells (Fig. 3a). Furthermore, Western blotting analysis showed that stable knockdown of CASC15 caused significant downregulation of β-catenin at the protein level in both A549 and H1299 cells, and this effect was largely abrogated by ectopic expression of SOX4 (Fig. 3b). However, CASC15 knockdown did not result in obvious change in β-catenin mRNA levels, regardless of whether or not SOX4 was overexpressed simultaneously (Fig. 3c), suggesting that β-catenin is not regulated by CASC15 or SOX4 at the transcriptional level in NSCLC cells. To investigate the effect of CASC15 on β-catenin protein stability, A549-shCASC15 cells and A549-shControl cells were treated with cycloheximide (CHX) to inhibit protein synthesis and harvested at the indicated time points. Results showed that the β-catenin protein levels decreased overtime in CHX-treated cells and CASC15 silencing significantly accelerated the degradation rate of β-catenin protein (Fig. 3d, upper panel). Meanwhile, ectopic expression of SOX4 in A549-shCASC15 cells extended the half-life of the β-catenin protein to that of A549-shControl cells (Fig. 3d, lower panel). Collectively, these results indicated that CASC15 activates Wnt signaling in NSCLC via SOX4-mediated stabilization of β-catenin protein.
CASC15 is transcriptionally activated by the hypoxia/HIF-1α signaling in NSCLC
Numerous studies have demonstrated that CASC15 expression is elevated in a variety of human malignancies, such as melanoma, gastric cancer, liver cancer, and acute leukemia[22, 27-29]. However, to date, little is known about the mechanisms governing CASC15 upregulation in cancer. To determine whether the hypoxia/HIF-1α signaling could activate CASC15 expression, NSCLC cells were placed in a 1% oxygen environment for 0~36 hr and the expression levels of HIF-1α and CASC15 were measured. The results showed that HIF-1α expression levels in A549 and H1299 cells were significantly induced upon hypoxia and reached an induction peak at ~12 hr (Fig. 4a, upper panel). Then, HIF-1α levels gradually decreased but remained elevated throughout the 36-hour period. Intriguingly, CASC15 expression started to increase after 12 hr of hypoxia and reached a 4~6-fold induction at 36 hr (Fig. 4a, lower panel). These results indicated that CASC15 might be transcriptionally regulated by the hypoxia/HIF-1α signaling in NSCLC cells. To test this hypothesis, we knocked down the expression of HIF-1α in A549 and H1299 cells under either normoxic or hypoxic conditions (Fig. 4b). Subsequent qPCR analysis revealed that hypoxia-induced upregulation of CASC15 was largely eliminated by HIF-1α silencing (Fig. 4c). Moreover, in vivo experiments demonstrated that stable knockdown of HIF-1α significantly decreased the expression level of CASC15 in A549 xenograft tissues (Fig. S4). Thus, we conclude that hypoxia induces CASC15 expression via a HIF-1α-dependent pathway.
To demonstrate whether CASC15 is directly regulated by HIF-1α, two adjacent putative hypoxia-response elements (HREs, also known as HIF-1 binding sites) were identified in the promoter of the CASC15 gene (Fig. 4d, left panel). Then, luciferase reporters driven by CASC15 promoter fragment with wild-type HREs (pGL3-CASC15) or mutated HREs (pGL3-CASC15-mut1, pGL3-CASC15-mut2, and pGL3-CASC15-mut1&2) were generated and tested under normoxic or hypoxic conditions (Fig. 4d, left panel). Under normoxic conditions, an approximately two-fold higher luciferase activity than that of the empty vector was detected in both pGL3-CASC15 and pGL3-CASC15-mut, as well as the VEGF-Luc reporter. However, under hypoxic conditions, a nearly six-fold induction of luciferase activity in pGL3-CASC15 construct was observed. Mutation of a single HRE in CASC15 promoter partially impaired the inductive effect caused by hypoxia, while mutation of both HREs decreased the luciferase activity to nearly basal levels (Fig. 4d, upper right panel). The VEGF-Luc positive control gave rise to a nine-fold induction of luciferase activity. These results strongly suggested that hypoxia-induced CASC15 transactivation is mainly dependent on intact HREs. To further confirm a HIF-1α dependent CASC15 transactivation, we knocked down the expression of HIF-1α and tested luciferase reporter activity in A549 cells under hypoxic conditions. Compared to the empty vector control, luciferase activity of pGL3-CASC15 construct under hypoxic conditions was 5-fold higher in A549-siControl cells, but less than 2.5-fold higher in A549-siHIF1A cells (Fig. 4d, lower right panel). Then, we performed ChIP assays to investigate whether HIF-1α transcription factor binds directly to the CASC15 promoter. The PCR-amplified fragment, corresponding to a portion of the CASC15 promoter where HIF-1α bound to, was detected in the input samples (Fig. 4e, upper panel, lanes 2 and 5) or in the sample immunoprecipitated with HIF-1α antibody only under hypoxic conditions (Fig. 4e, upper panel, lane 7), but not under normoxic conditions (Fig. 4e, upper panel, lane 4). The HIF-1α binding to the VEGF-A promoter, serving as positive control, was shown (Fig. 4e, lower panel). Altogether, these results demonstrated that CASC15 is transcriptionally activated by the hypoxia/HIF-1α signaling in NSCLC.
HIF-1α/CASC15/SOX4/β-catenin pathway is activated in a substantial subset of NSCLC patients
Based on the above evidence, we concluded that lncRNA CASC15 could exert tumor-promoting effects in NSCLC cell lines through a novel HIF-1α/CASC15/SOX4/β-catenin axis. To validate this axis in clinical samples, we downloaded and analyzed the gene expression profiles of HIF1A, CASC15, SOX4, and CTNNB1 (β-catenin) in two lung adenocarcinoma cohorts from TCGA database. The first cohort contains 58 matched pairs of lung adenocarcinoma and adjacent normal lung tissues, and the results showed that all four genes involved in the HIF-1α/CASC15/SOX4/β-catenin axis are highly expressed in cancer tissues compared with normal tissues (Fig. 5a). Then, we performed co-expression analysis in another cohort containing 515 lung adenocarcinoma tissues. We found that positive correlations are particularly strong between HIF1A and CASC15, CASC15 and SOX4, and SOX4 and CTNNB1 (Fig. 5b), indicating potential regulatory relationships between the genes involved in the HIF-1α/CASC15/SOX4/β-catenin pathway.
Next, we performed RNA-ISH and IHC assays to examine the expression levels of lncRNA CASC15 and its related proteins, in A549 xenograft tissues (refer to Fig. 1g) and NSCLC tissue microarrays. As shown in Fig. 6a, HIF-1α, CASC15, SOX4, and β-catenin were aberrantly overexpressed in a substantial proportion of NSCLC patients (45.7%, 16/35), while in normal lung tissues their expressions were nearly absent. Intriguingly, we found that silencing of CASC15 in A549 xenograft tissues remarkably reduced the protein levels of SOX4 and β-catenin, but had no effect on HIF-1α expression (Fig. 6c), supporting our notion that CASC15 functions at an intermediate node in the HIF-1α/CASC15/SOX4/β-catenin signaling axis (Fig. 6d). Furthermore, we examined the expression levels of CASC15 and related proteins in fresh samples from seven lung adenocarcinomas and three normal lung tissues. qPCR and Western blotting analyses showed significant positive correlations between levels of CASC15 and its regulators/targets (Fig. 6b). Taken together, these data demonstrated that the HIF-1α/CASC15/SOX4/β-catenin pathway was activated in a substantial subset of NSCLC patients.