Hypoxic Microenvironment-Induced Reduction in PTEN-L Secretion Promotes Non-Small Cell Lung Cancer Metastasis through PI3K/AKT Pathway

Abstract

Objective: Lung cancer is the leading cause of cancer-related deaths worldwide. The aim of this study was to investigate the effects of hypoxic microenvironment on PTEN-L secretion and the effects of PTEN-L on the metastasis of non-small cell lung cancer (NSCLC) and the potential mechanisms.

Methods: The expression levels of PTEN-L in NSCLC tissues, cells, and cell culture media were detected. The transfection of PTEN-L overexpression construct or HIF-1α-siRNAs was conducted to manipulate the expression of PTEN-L or HIF-1α. NSCLC cells were introduced into 200 μM CoCl2 medium for 72 hours under 37°C to simulate hypoxia. The proliferation and apoptosis of the A549 cells were determined by the Cell Counting Kit-8 assay and Annexin V-FITC/PI-stained flow cytometry assay, respectively. Wound healing assay and transwell invasion assay were used to measure the migration and invasion of A549 cells. The protein expression of PTEN, PTEN-L, PI3K/AKT pathway-related proteins, and HIF-1α was detected by Western blot.

Results: PTEN and PTEN-L are downregulated in lung cancer tissues and cells. The protein expression of PTEN-L in the culture medium of lung cancer cell lines is decreased. The hypoxic microenvironment inhibits PTEN-L secretion. The low level of PTEN-L promotes cell proliferation, migration, and invasion, as well as inhibits apoptosis of A549 cells. The overexpression of PTEN-L attenuated the activation of the PI3K/AKT pathway by the hypoxic microenvironment. The knockdown of HIF-1α upregulates PTEN-L secretion under hypoxia.

Conclusions: The hypoxic microenvironment inhibits PTEN-L secretion and thus activates PI3K/AKT pathway to induce proliferation, migration, and invasion promotion, and apoptosis inhibition in NSCLC cells.

Figure 1

A decrease in PTEN and PTEN-L expression was observed in NSCLC tissues and cells. A, B, The relative protein expression of PTEN and PTEN-L in NSCLC tissues and para-carcinoma tissues (control) was examined by Western blot. C, D, The relative protein expression of PTEN and PTEN-L in normal lung epithelial cells (16HBE) and lung cancer cell lines (16HBE, A549, H226, H460, SPC-A1) was examined by Western blot. E, F, The relative protein expression of PTEN-L in the culture medium of normal lung epithelial cells (16HBE) and lung cancer cell lines (16HBE, A549, H226, H460, SPC-A1) was examined by Western blot. (^∗∗∗p < 0.001 vs. control group or 16HBE group).

Figure 2

Hypoxic microenvironment inhibits PTEN-L secretion to promote proliferation and inhibit apoptosis of A549 cells. A, B, The effect of hypoxia on PTEN and PTEN-L protein expression was detected by Western blot. C, D, The relative protein expression of PTEN-L in A549 cell cultures under hypoxic and/or overexpressing PTEN-L was examined by Western blot. E The relative proliferation viability of A549 cells under hypoxic and/or overexpressing PTEN-L was examined by CCK-8 assay. F, G, Apoptosis of A549 cells under hypoxic and/or overexpressing PTEN-L was examined by double staining with Annexin V-FITC/propidium iodide (PI) and flow cytometry. (^∗∗p < 0.01,^∗∗∗p < 0.001 vs. control group; ^∧∧p < 0.01 vs. hypoxia group; ^##p < 0.01 vs. PTEN-L group).

Figure 3

Hypoxic microenvironment inhibits PTEN-L secretion to promote migration and invasion of A549 cells and activate PI3K/AKT signaling. A, B, Wound healing assay to determine migration of A549 cells. C, D, Transwell invasion assay was performed to determine the invasion of A549 cells. E, F, Expression of PI3K/AKT pathway-related proteins was detected by Western blot. (^∗∗∗p < 0.001 vs. control group; ^∧p < 0.05,^∧∧p < 0.01 vs. hypoxia group; ^##p < 0.01,^###p < 0.001 vs. PTEN-L group).

Figure 4

Silencing HIF-1α significantly reverses the decrease in PTEN-L and PTEN secretion-induced hypoxic environment. A, B, Relative protein expression of HIF-1α, PTEN, and PTEN-L was detected by Western blot in A549 cells. C, D, Transfection effect of si-HIF-1α was detected by Western blot in A549 cells. E, F, Relative protein expression of PTEN and PTEN-L was detected by Western blot in A549 cells. G, H, Relative protein expression of PTEN-L was detected by Western blot in the medium of A549 cells. (^∗∗p < 0.01,^∗∗∗p < 0.001 vs. control group; ^∧∧p < 0.01 vs. hypoxia group; ^##p < 0.01,^###p < 0.001 vs. si-HIF-1α group).