JAG1 Is Associated with Poor Survival through Inducing Metastasis in Lung Cancer

Abstract

JAG1 is a Notch ligand that plays a critical role in multiple signaling pathways. However, the functionality of JAG1 in non-small cell lung cancer (NSCLC) has not been investigated thoroughly. By comparison of gene transcripted RNA profiles in the cell line pair with differential invasion ability, we identified JAG1 as a potential metastasis enhancer in lung cancer. Ectopic expression of JAG1 on lung cancer cells enhanced cell migration and invasion as well as metastasis in vitro and in vivo. Conversely, knockdown of JAG1 with siRNA in highly invasive cancer cells led to the reduction of migration and invasion. In clinical analysis, JAG1 mRNA expression was higher in tumors than in adjacent normal tissues in 14 of 20 patients with squamous cell carcinoma (SCC). SCC patients with higher JAG1 transcription had poor overall survival than those with low-transcripted JAG1. Microarray analysis indicated that the enforced JAG1 transcription was associated with an elevated HSPA2 RNA transcription, which played a role in promoting cancer cell migration and invasion. In conclusion, this is the first study that demonstrated that JAG1 might act as a potential prognostic marker and JAG1/HSPA2 axis mediates lung cancer malignancy at least partly.

Fig 1. JAG1 acts as an invasion-promoting gene.

(A) Identification of JAG1 in an invasion model of lung cancer cells. JAG1 is up-regulated in highly invasive lung cancer cells. Left, JAG1 mRNA level was assessed by oligonucleotide microarray analysis. Middle, JAG1 mRNA level was measured by real-time quantitative RT-PCR. The experiments were performed in triplicate and data were represented as mean ± SD. Right, JAG1 protein expression was evaluated by Western blot. β-actin was used as an internal control. (B) JAG1 promotes cell migration and invasiveness in vitro. Left, JAG1 was stably overexpressed in CL1-0 cells. JAG1 mRNA was measured by real-time quantitative RT-PCR in triplicate. Data were represented as mean ± SD. Middle and Right, migration and invasion ability of CL1-0 cells with constitutive JAG1 expression and mock control cells were evaluated by migration assay and matrigel invasion assay. Data presented as mean ± SD of three experiments. *: p < 0.05 as compared to the mock group. (C) Knockdown of JAG1 inhibits cell migration and invasiveness in vitro. Left, JAG1 mRNA was knocked down by siRNAs in CL1-5 cells assayed by real-time quantitative RT-PCR in triplicate. Two different siRNAs (siJAG1-1 and siJAG1-2) were used to silence JAG1. NC, negative control. Middle and Right, migration and invasion capability of JAG1-silencing transfectants were analyzed by migration and matrigel invasion assays. Data shown as mean ± SD; *: p < 0.05 compared with the negative control.

Fig 2. JAG1 enhances migration and invasion in different lung cancer cell lines and promotes metastasis in vivo.

(A) JAG1 promotes cell migration and invasiveness in vitro. Left, JAG1 was transiently overexpressed in A549 and H226 cells. JAG1 mRNA was measured by real-time quantitative RT-PCR and normalized to TBP in triplicate. Middle and Right, migration ability and invasiveness of cells with transient JAG1 overexpression and control cells were evaluated by migration and matrigel invasion assays. *: p < 0.05, compared with mock. Data were represented as mean ± SD in triplicate. (B) JAG1 promotes metastasis in vivo. Left, number of nodules in SCID mice. The Mock CL1-0 cells and JAG1-overexpressing cell lines were inoculated into severe combined immunodeficiency (SCID) mice by tail vein injection. After 10 weeks, mice were sacrificed. Number of tumors derived from mock and JAG1 transfectants was measured under the dissection microscope. Data were represented as mean ± SD (n = 10 in mock control group and n = 13 in JAG1 transfectant group). *: p < 0.05, compared with mock control. Right, appearances of the lungs from mice injected with CL1-0 mock and JAG1 transfectants and the representative H&E staining sections of the lungs from mice. Red arrow heads indicate metastatic tumor nodules in lungs. Black arrows indicate the micrometastasis of lung cancer cells.

Fig 3. JAG1 expression and NSCLC patients’ survival.

(A and B) JAG1 mRNA expression in tumor and normal parts of lung cancer. JAG1 mRNA in tumors and adjacent normal tissues of NSCLC (A) and subtype squamous carcinoma patients (B) was assessed by real-time quantitative RT-PCR and normalized to TBP. (C) JAG1 mRNA level and overall survival of subtype squamous carcinoma patients. All statistical tests were two sided, and p <0.05 was considered to be statistically significant.

Fig 4. HSPA2 mRNA expression is regulated by JAG1.

(A and B) JAG1 was transiently overexpressed in A549 and H226 (A) or knockdown by JAG1 siRNAs in CL1-5 (B). HSPA2 mRNA was measured by real-time quantitative RT-PCR and normalized to TBP. (C) Migration ability of CL1-0 cells with transient HSPA2 overexpression and control cells was evaluated by migration assay. *, p <0.05, compared with mock control. Data were represented as mean ± SD (n = 3 per group). (D) Invasiveness of CL1-0 cells with transient HSPA2 overexpression and control cells as evaluated by matrigel invasion assay. *, p <0.05, compared with mock control. Data were represented as mean ± SD (n = 3 per group).

Fig 5. Proteolytic status of NOTCH family or downstream molecules changes in JAG1 manipulated lung cancer cell lines.

Cell was transiently overexpressed JAG1 by expression plasmid or silenced JAG1 by siRNA strategy followed by immunoblotted with NICD1, NICD2, NICD3, and NICD4 antibodies or real-time quantitative PCR for NOTCH downstream molecules mRNA level analysis. (A) JAG1 overexpressed in CL1-0 cells followed by Western blot analysis for NICDs proteolytic status or real-time quantitative PCR for NOTCH downstream molecules mRNA level analysis. (B) JAG1 overexpressed in H1299 and H838 cell lines followed by Western blot analysis for NICDs proteolytic status. (C) JAG1 silenced in HOP62 and H322M cell lines followed by Western blot analysis for NICDs proteolytic status. DAPT is a gamma-secretase inhibitor and used for NOTCH signaling control. UD, undetermined due to undetectable expression level; *, p <0.05 compared with mock control. Data were represented as mean ± SD (n = 3 per group).