FAM83A signaling induces epithelial-mesenchymal transition by the PI3K/AKT/Snail pathway in NSCLC

Abstract

Family with sequence similarity 83, member A (FAM83A), as a potential tumor promoter, was reported to contribute to the progression of several malignant tumors. However, the significance of FAM83A in invasion and metastasis of non-small cell lung cancer (NSCLC) remains largely unknown. In this study, we found that FAM83A expression was significantly increased in NSCLC tissues. High expression of FAM83A was positively associated with tumor metastasis and poor survival of NSCLC patients. Functional experiments revealed that FAM83A knockdown could suppress NSCLC cell migration and invasion both in vivo and in vitro. While opposite results were observed in FAM83A-transfected cells. Mechanically, we found that FAM83A promoted NSCLC cell migration and invasion by inducing epithelial-mesenchymal transition (EMT) via PI3K/ATK/Snail signaling. Rescue experiment demonstrated that inhibition of either AKT or Snail could partially counteract the promoting effect of FAM83A overexpression in NSCLC metastasis. Taken together, our findings are the first time to demonstrate that increased expression of FAM83A in NSCLC was correlated with EMT and tumor metastasis, which may provide a novel therapeutic target in NSCLC treatment.

Keywords: EMT; FAM83A; non-small cell lung cancer.

Figure 1

FAM83A was overexpression in NSCLC tissues and correlated with more aggressive clinical characteristics in the TCGA dataset. (A) Heatmaps showing the clustering patterns of differentially expressed FAM83A between normal and lung cancer specimens. (B) Box plot analysis of the FAM83A mRNA levels in lung adenocarcinoma (LUAD) and human lung squamous cell carcinoma (LUSC) tissue samples. (C–D) Kaplan-Meier plots of overall-survival (C) and disease-free survival (D) in NSCLC patients with high and low levels of FAM83A. The dotted line indicates the 95% confidence interval. (E) Pearson’s test showed that FAM83A had weak positive correlation with Snail (r = 0.17, P<0.001) in NSCLC tissues.

Figure 2

FAM83A was highly expressed in NSCLC tissues and correlated with worse survival. (A) Immunohistochemical staining showed that FAM83A was highly expressed in NSCLC tumors (n = 49/101) compared with normal lung tissues (n = 2/50, P < 0.05). (B–C) Kaplan-Meier plots of overall-survival (B) and progression-free survival (C) in NSCLC patients with high and low levels of FAM83A. Scale bar, 100 μm.

Figure 3

Suppression of FAM83A inhibited NSCLC cell migration and invasion capacity in vitro. (A) FAM83A protein level was assessed in NSCLC cell lines using Western blot analysis. β-actin was used as a loading control. (B) The intensity of protein levels was quantified using Image Lab 5.2.1 software and normalized to β-actin. (C–G) H661 and H1299 cells were transduced with FAM83A lentivirus (LV-shFAM83A-1/2) and a scrambled shRNA lentivirus (LV-shRNA-NC). The suppression of FAM83A in H661 and H1299 cells was confirmed at both the protein (C–F) and RNA (G) levels. (H–I) H661-shRNA-NC, H661-shFAM83A-1, H1299-shRNA-NC, and H1299-shFAM83A-1 cells were subjected to a wound-healing assay. Images were taken at 0 h and 24 h. (J–K) Transwell assays assessed tumor cell migration and invasion capacity in FAM83A-knockdown H661 and H1299 cell lines. Scale bar, 200 μm. Error bars: mean ± SD (n=3). *p<0.05, **p<0.01, and ***p<0.001 were considered to indicate a statistically significant difference.

Figure 4

Overexpression of FAM83A facilitated NSCLC cell migration and invasion capacity in vitro. H460 and A549 cells were transduced with a FAM83A lentivirus (LV-FAM83A) or a control lentivirus (LV-NC). (A–E) Overexpression of FAM83A in H460 and A549 cells was confirmed at both the protein (A–D) and RNA (E) levels. (F–G) H460 and A549 cells transduced with the FAM83A lentivirus were subjected to a wound-healing assay. Images were taken at 0 h and 24 h. (H–I) Transwell assays assessed tumor cell migration and invasion capacity. Scale bar, 200 μm. Error bars: mean ± SD (n=3). *p<0.05, **p<0.01, and ***p<0.001 were considered to indicate a statistically significant difference.

Figure 5

FAM83A facilitated NSCLC cell EMT progression. (A–D) Protein levels of FAM83A, Vimentin, E-cadherin, Snail, and Twist were detected by Western blot in stable H1299-LV-shRNA-NC, H1299-LV-shFAM83A-1, A549-LV-NC and A549-LV-FAM83A cells. β-actin was used as a loading control. (E–F) Immunofluorescence of Vimentin and E-cadherin in A549 and H1299 cells after manipulation of FAM83A expression. The nucleus was counterstained with DAPI. The fourth panel of each shows the merged image of the previous panels. Scale bar, 100 μm. Error bars: mean ± SD (n=3). NS, no significant, *p<0.05, **p<0.01, and ***p<0.001 were considered to indicate a statistically significant difference.

Figure 6

FAM83A regulated NSCLC cell EMT progression through the PI3K/AKT/Snail pathway. (A–C) Protein levels of PI3K, P-PI3K p85 subunit (p-Y458), AKT, and P-AKT (p-S473) were detected by Western blot in stable H1299-LV-shRNA-NC, H1299-LV-shFAM83A-1, A549-LV-NC and A549-LV-FAM83A cells. β-actin was used as a loading control for PI3K and AKT. (D–E) H460 and A549 with stable FAM83A overexpression cells were transduced with Snail siRNAs (siSnail-1/2) and a scrambled siRNA (Vector). The suppression of Snail in those cells was confirmed by Western blot. (F–G) FAM83A-transfected H460 and A549 cells (with or without inhibition of MK2206 or Snail) were subjected to a wound-healing assay. Images were taken at 0 h and 24 h. (H–I) Transwell assay was used to evaluate the effects of MK2206 and Snail knockdown on cell migration and invasion. Scale bar, 200 μm. Error bars: mean ± SD (n=3). *p<0.05, **p<0.01, and ***p<0.001 were considered to indicate a statistically significant difference.

Figure 7

Inhibition of AKT impaired the EMT phenotype owing to FAM83A overexpression. (A) Immunofluorescence of Vimentin and E-cadherin in FAM83A-transfected A549 cells with or without MK2206. The nucleus was counterstained with DAPI. The fourth panel of each shows the merged image of the previous panels. (B–C) Protein levels of Vimentin, E-cadherin, Snail, AKT and P-AKT (p-S473) were detected by Western blot in stable A549-LV-NC and A549-LV-FAM83A cells (with or without MK2206). Scale bar, 100 μm. Error bars: mean ± SD (n=3). NS, no significant, *p<0.05, **p<0.01, and ***p<0.001 were considered to indicate a statistically significant difference.

Figure 8

Increased FAM83A expression promoted lung metastasis in vivo, and inhibition of AKT reduced the metastatic foci owing to FAM83A overexpression. (A) Schematic diagram of the metastasis model in mice. (B) Stable H1299-LV-shFAM83A-1 or A549-LV-FAM83A cells (each also expressing luciferase) were transplanted into nude mice (tail vein injection). Two groups of nude mice overexpressing FAM83A were then treated with 30% Captisol diluents (Vector) or MK2206 at a dose of 50 mg/kg three times a week. Tumor formation in the lungs and distant metastasis were monitored by bioluminescence imaging. (C) Representative images and summary of the number of lung metastatic nodules. Error bars: mean ± SD (n=3). *p<0.05, **p<0.01, and ***p<0.001 were considered to indicate a statistically significant difference.

Figure 9

Increased FAM83A expression promoted lung metastasis and EMT in vivo, and AKT inhibitor reduced EMT owing to FAM83A overexpression. (A) HE staining of lung tissues in several groups (A549 and H1299 cells after manipulation of FAM83A expression with or without MK2206) of tumor-bearing mice. (B) Immunohistochemical staining for FAM83A, E-cadherin and Vimentin was performed using serial sections of mouse lung tissues. Scale bar, 100 μm.