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. 2018 Nov;25(11):1980-1995.
doi: 10.1038/s41418-018-0084-9. Epub 2018 Mar 6.

LncRNA CASC9 Promotes Esophageal Squamous Cell Carcinoma Metastasis Through Upregulating LAMC2 Expression by Interacting With the CREB-binding Protein

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LncRNA CASC9 Promotes Esophageal Squamous Cell Carcinoma Metastasis Through Upregulating LAMC2 Expression by Interacting With the CREB-binding Protein

Yan Liang et al. Cell Death Differ. .
Free PMC article

Abstract

Esophageal squamous cell carcinoma (ESCC) is the main subtype of esophageal cancer. Long noncoding RNAs (lncRNAs) are thought to play a critical role in cancer development. Recently, lncRNA CASC9 was shown to be dysregulated in many cancer types, but the mechanisms whereby this occurs remain largely unknown. In this study, we found that CASC9 was significantly upregulated in ESCC tissues, with further analysis revealing that elevated CASC9 expression was associated with ESCC prognosis and metastasis. Furthermore, we found that CASC9 knockdown significantly repressed ESCC migration and invasion in vitro and metastasis in nude mice in vivo. A microarray analysis and mechanical experiments indicated that CASC9 preferentially affected gene expression linked to ECM-integrin interactions, including LAMC2, an upstream inducer of the integrin pathway. We demonstrated that LAMC2 was consistently upregulated in ESCC and promoted ESCC metastasis. LAMC2 overexpression partially compromised the decrease of cell migration and invasion capacity in CASC9 knockdowns. In addition, we found that both CASC9 and LAMC2 depletion reduced the phosphorylation of FAK, PI3K, and Akt, which are downstream effectors of the integrin pathway. Moreover, the reduction in phosphorylation caused by CASC9 depletion was rescued by LAMC2 overexpression, further confirming that CASC9 exerts a pro-metastatic role through LAMC2. Mechanistically, RNA pull-down and RNA-binding protein immunoprecipitation (RIP) assay indicated that CASC9 could bind with the transcriptional coactivator CREB-binding protein (CBP) in the nucleus. Chromatin immunoprecipitation (ChIP) assay additionally illustrated that CASC9 increased the enrichment of CBP and H3K27 acetylation in the LAMC2 promoter, thereby upregulating LAMC2 expression. In conclusion, we demonstrate that CASC9 upregulates LAMC2 expression by binding with CBP and modifying histone acetylation. Our research reveals the prognostic and pro-metastatic roles for CASC9 in ESCC, suggesting that CASC9 could serve as a biomarker for prognosis and a target for metastasis treatment.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
CASC9 overexpression in ESCC tissues correlates with ESCC aggressiveness. a Fold change in CASC9 expression between ESCC tissues and normal tissues normalized by log2 using RT-qPCR. b The correlation between CASC9 expression and tumor differentiation grade: normal, well, moderate, and poor. c The correlation between CASC9 expression and primary tumor invasion depth. T1: mucous layer, T2: muscularis propria layer, T3/T4: the adventitia layer and surrounding tissues. d The correlation between CASC9 expression and lymph node metastasis. Negative: no lymph node metastasis, positive: with lymph node metastasis. e The correlation between CASC9 expression and TNM stage. Stage I, stage II, and stage III represent different TNM stage of ESCC according to the latest AJCC (American Journal of Critical Care) guide. f High CASC9 expression in tumor tissues was associated with reduced survival of ESCC patients, as analyzed by Kaplan–Meier method. g High T:N expression ratio of CASC9 was associated with reduced survival. T:N, CASC9 expression ratio between tumor and adjacent normal tissue. h No significant association between CASC9 expression and survival was detected in normal tissues. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 2
Fig. 2
CASC9 promotes ESCC migration and invasion in vitro. a, b Transwell assays were performed in KYSE150 and KYSE450 cells transfected with two different siRNAs. The number of cells that migrated or invaded were counted in three different fields. Original magnification, ×200. Scale bars, 200 µm. c Transwell assays were performed in EC109 cells transfected with different vectors. Original magnification, ×200. Scale bars, 200 µm. d Wound healing assay in KYSE150 and KYSE450 cells transfected with two siRNAs. Original magnification, ×100. Scale bars, 200 µm. The distance of wound healing was measured and calculated as a percentage of the distance at 0 h. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3
CASC9 promotes ESCC metastasis in vivo. a Representative images of mice from different treatment groups 8 weeks after tumor injections. Three mice from the LV-NC group presented bone and other remote metastasis. The metastatic tumors were detected under a stereoscope. Scale bars, 1 cm. b Number of metastatic nodules in lungs and other organs in mice from LV-CASC9 and LV-NC groups. c Bar chart of lung metastasis nodules in LV-CASC9 and LV-NC groups. d Representative images of lung metastases under stereoscope. Original magnification, ×1.5. Scale bars, 1 cm. e Representative images of HE and IHC staining of lung metastasis. Original magnification, ×400. Scale bars, 100 µm. f Signaling pathways of downregulated genes after CASC9 knockdown in KYSE450 cells detected by microarray analysis. g, h, i Gene expression of ECM–receptor interactions and focal adhesion pathways after CASC9 knockdown or overexpression detected by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4
CASC9 upregulates the expression of genes associated with ECM–receptor interactions and focal adhesion pathways. a, b Western blot and IF indicates the expression of LAMC2 and ITGB4 after CASC9 knockdown using two siRNAs in KYSE150 and KYSE450 cells. Original magnification, ×400. Scale bars, 40 µm. c Fold change in LAMC2 expression between ESCC tissues and normal tissues normalized by log2 using RT-qPCR. d The correlation between LAMC2 expression and primary tumor invasion depth. T1/T2: mucous layer and muscularis propria layer, T3/T4: the adventitia layer and surrounding tissues. e, f ESCC patients with lymph node metastasis and advanced TNM stage showed higher LAMC2 expression. Negative: no lymph node metastasis, positive: with lymph node metastasis. g Kaplan–Meier analysis revealed that high LAMC2 expression predicted poor prognosis in ESCC. h Correlation analysis of LAMC2 and CASC9 expression in ESCC tissues by RT-qPCR. Axis values are transformed by −Log2 of CASC9 expression. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 5
Fig. 5
CASC9 promotes ESCC metastasis through regulation of LAMC2 expression. a Transwell assays showed that LAMC2 knockdown attenuated the migration and invasion abilities of KYSE150 and KYSE450 cells. Original magnification, ×100. Scale bars, 200 µm. b Transwell assays showed that LAMC2 overexpression enhanced the migration and invasion abilities of KYSE150 and KYSE450 cells. Original magnification, ×100. Scale bars, 200 µm. c The rescue experiment with Transwell assays was performed in KYSE150 and KYSE450 cells co-transfected with si-NC or si-CASC9-2 and pcDNA3.1 or pcLAMC2. Original magnification, ×100. Scale bars, 200 µm. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 6
Fig. 6
CASC9 activates FAK-PI3K/Akt signaling pathways through LAMC2. a Western blot analysis showed altered levels of pFAK, pPI3K, and pAkt after CASC9 or LAMC2 knockdown in KYSE150 and KYSE450 cells. b Western blot analysis of FAK-PI3K/Akt signaling pathways of the rescue experiment. c RT-qPCR analysis of MMP10 and MMP13 expression. d, e The expression of MMP10 and MMP13 in KYSE150 and KYSE450 cells after CASC9 knockdown. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 7
Fig. 7
CASC9 upregulates LAMC2 expression through interacting with CBP. a Subcellular localization of CASC9 and U6 in KYSE150 and KYSE450 detected by RNA FISH. Original magnification, ×630. Scale bars, 20 µm. b, c RT-qPCR and western blot analysis of LAMC2 expression after CBP knockdown in KYSE150 and KYSE450. d Luciferase reporter assay was performed using vector-containing sequences of the LAMC2 promoter region. e RNA pull-down assay showed that CASC9 could retrieve CBP in KYSE150 and KYSE450 cells as detected by western blot. Pull-down indicates the retrieved protein. FT represents the flow-through protein after RNA–protein binding reaction. Input indicates the total cell protein used. f RIP experiment showed that anti-CBP antibody could precipitate CASC9 in KYSE150 and KYSE450 cells. g, h ChIP-qPCR revealed that CASC9 knockdown decreased the enrichment of CBP and the level of H3K27ac acetylation at the promoter region of LAMC2. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 8
Fig. 8
Model of CASC9 interaction with CBP and the signaling pathways involved in pro-metastatic function. a In the nucleus, CASC9 recruits CBP and modulates H3K27ac levels at the LAMC2 promoter, thereby facilitating gene transcription. b In the cytoplasm, LAMC2 is exported to the extracellular matrix and interacts with ITGB4 to activate FAK-PI3K/Akt signaling pathways, thereby promoting ESCC metastasis

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