SPANXA suppresses EMT by inhibiting c-JUN/SNAI2 signaling in lung adenocarcinoma

Abstract

SPANXA (Sperm Protein Associated with the Nucleus on the X-chromosome, family members A1/A2) acts as a cancer-testis antigen expressed in normal testes, but dysregulated in various tumors. We found that SPANXA is highly expressed in low-invasive CL1-0 cells compared with isogenous high-invasive CL1-5 cells. SPANXA was preferably expressed in tumor tissues and associated with the prolonged survival of lung adenocarcinomas. SPANXA suppressed the invasion and metastasis of lung cancer cells in vitro and in vivo. By the expression microarray and pathway analysis, we found that the SPANXA-altered genes were enriched in the epithelial-mesenchymal transition (EMT) pathway. SPANXA reduced SNAI2 expression resulted in up-regulating E-cadherin. c-JUN acts as the positive-regulator of EMT. Silencing SPANXA increased c-JUN mRNA expression and blockage of c-JUN led to SNAI2 down-regulation. Our results clearly characterized SPANXA as an EMT inhibitor by suppressing c-JUN-SNAI2 axis in lung adenocarcinoma.

Keywords: AP-1; E-cadherin; SLUG; SPANX family; metastasis.

Figure 1. SPANXA suppresses cell migration and invasion

(A) Establishment of SPANXA-expressing cells in highly invasive cells, CL1-5, and in low SPANXA-expressing cells, EKVX. The SPANXA expression was detected by anti-V5. (B) (C) Cell migration and invasion abilities of mock or SPANXA-expressing cells measured by Transwell assay. (D) Cell motility rate of SPANXA-expressing cells and mock control were measured by single cell tracking. All experiments were performed in triplicates. *P < 0.05 (mean ± SD, n = 3).

Figure 2. Knockdown of SPANXA increases cell migration and invasion in vitro

(A) Cell migration and invasion abilities of silencing SPANXA in stably SPANXA-expressing CL1-5 cells. (B) SPANXA expression in SPANXA-silencing CL1-0 and H1437 cells assessed by qRT-PCR. (C) Cell migration and invasion abilities of SPANXA-silencing CL1-0 and H1437 cells. All experiments were performed in triplicates. *P < 0.05 (mean ± SD, n = 3).

Figure 3. SPANXA attenuates metastasis in vivo

(A) In vivo tumor metastasis effect of SPANXA was analyzed by an experimental metastasis assay with stably SPANXA-expressing CL1-5 cells and mock control cells, which were intravenously injected into NOD-SCID mice. The metastatic tumor nodules were calculated. *P < 0.05. (B) Image of lung surface nodules. Anterior lungs showed on the upper part and posterior lungs on the lower part. Scale bar, 0.5 μm.

Figure 4. SPANXA inhibits EMT process

(A) Cell morphology of stably SPANXA-expressing CL1-5 cells and mock control cells. (B) Anti-V5 and anti-F-actin immunofluorescence staining of mock control and stably SPANXA-expressing CL1-5 cells. (C) Expression of EMT marker in stably SPANXA-expressing CL1-5 cells was assessed by immunoblot assays. All experiments were performed in duplicates.

Figure 5. SPANXA represses cell invasion through deregulating SNAI2

(A) Protein and mRNA expressions of SNAI2 in stably SPANXA-expressing CL1-5 cells and mock control cells assessed by qRT-PCR and Western blot assays. *P < 0.05 (mean ± SD, n = 3). (B) RNA expression of SNAI2 in SPANXA-silencing CL1-0 cells and shLuc control cells assayed by qRT-PCR. *P < 0.05 (mean ± SD, n = 3). (C) The stably SPANXA-expressing CL1-5 cells were infected with shSPANXA lentivirus and assayed for SNAI2 protein expression by Western blot assays. The experiment was performed in duplicates. (D) Cell invasion and expression of SNAI2 and E-cadherin of stably SPANXA-expressing CL1-5 cells was assessed in presence of SNAI2 overexpression. *P < 0.05 (mean ± SD, n = 3) (E) Cell invasion of stably SPANXA-silencing CL1-0 cells was assessed under SNAI2 silencing. The lower row indicated SNAI2 RNA expression assayed qRT-PCR. *P < 0.05 (mean ± SD, n = 3). All assays were performed in two independent experiments.

Figure 6. SPANXA suppresses SNAI2 expression and EMT through repressing c-JUN

(A) The different ratios of c-JUN and c-FOS were ectopically introduced into stably SPANXA-expressing CL1-5 cells. The SNAI2 and E-cadherin expressions were measured by qRT-PCR. *P < 0.05 (mean ± SD, n = 3). (B) Knockdown of c-JUN in stably SPANXA-silencing CL1-0 cells reduced the SNAI2 expression. The lower row indicated the c-JUN expression. *P < 0.05 (mean ± SD, n = 3). (C) Model of SPANXA-regulating network. SPANXA inhibits the c-JUN/SNAI2/CDH1 signalling and EMT process. The dotted line indicates the unexplored part of SPANXA regulation. All assays were performed in two independent experiments.