Alkaline phosphatase downregulation promotes lung adenocarcinoma metastasis via the c-Myc/RhoA axis

Abstract

Background: Lung adenocarcinoma (LUAD) metastasis significantly reduces patient survival; hence inhibiting the metastatic ability of lung cancer cells will greatly prolong patient survival. Alkaline phosphatase (ALPL), a homodimeric cell surface phosphohydrolase, is reported to play a controversial role in prostate cancer and ovarian cancer cell migration; however, the function of ALPL in LUAD and the related mechanisms remain unclear.

Methods: TCGA database was used to analysis the expression of ALPL, and further verification was performed in a cohort of 36 LUAD samples by qPCR and western blot. Soft-agar assay, transwell assay and lung metastasis assay were employed to detect the function of ALPL in LUAD progression. The qPCR, luciferase promoter reporter assay and western blot were used to clarify the molecular mechanisms of ALPL in promoting metastasis in LUAD.

Results: ALPL was downregulated in LUAD, and the disease-free survival rate of patients with low ALPL was significantly reduced. Further studies showed that overexpression of ALPL in LUAD cell lines did not significantly affect cell proliferation, but it did significantly attenuate lung metastasis in a mouse model. ALPL downregulation in LUAD led to a decrease in the amount of phosphorylated (p)-ERK. Because p-ERK promotes the classical c-Myc degradation pathway, the decrease in p-ERK led to the accumulation of c-Myc and therefore to an increase in RhoA transcription, which enhanced LUAD cell metastasis.

Conclusion: ALPL specially inhibits the metastasis of LUAD cells by affecting the p-ERK/c-Myc/RhoA axis, providing a theoretical basis for the targeted therapy of clinical LUAD.

Keywords: ALPL; C-Myc degradation; Lung adenocarcinoma (LUAD); Metastasis; RhoA.

Fig. 1

Alkaline phosphatase (ALPL) is downregulated in LUAD. a ALPL mRNA expression was analyzed in the 58 paired LUAD cases in the Cancer Genome Atlas (TCGA). b ALPL mRNA expression was analyzed in 36 paired fresh clinical LUAD tissues. c Western blot analysis of total protein lysates prepared from 36 paired fresh clinical LUAD tissues. Then perform grayscale analysis and statistics. d The relationship between ALPL expression in LUAD patients in the TCGA database and the disease-free survival rate. e Western blot analysis of ALPL protein levels in LUAD cell lines. GAPDH was used as an internal control. All results were presented as the mean ± SD and analyzed by Student’s t-test. The survival curve was analyzed by log-rank test. Asterisks (*) represent statistical significance (P < 0.05)

Fig. 2

Alkaline phosphatase (ALPL) has no effect on the proliferation of LUAD cells. Western blot analysis of A549 (a) and HCC827 (b) cells stably transfected with an ALPL overexpression plasmid or a control plasmid. GAPDH was used as an internal control. Soft-agar assay to determine the effect of ALPL overexpression on anchorage-independent growth of A549 (c, d) and HCC827 (e, f) cells. Representative images of colonies were captured by microscopy after 3 weeks of incubation (c, e), Scale bars: 200 µm. The number of colonies with > 32 cells were counted in A549 (Vector) and A549 (ALPL) cells (d), and in HCC827 (Vector) and HCC827 (ALPL) cells (f). The results represent the number of colonies per 10,000 cells. Data were presented as the mean ± SD and analyzed by Student′s t-test, n = 3. ns, no statistical significance relative to the vector control cells (P > 0.05)

Fig. 3

Alkaline phosphatase (ALPL) inhibited the metastasis of LUAD cells in vivo and in vitro. Transwell assays were performed to determine the effect of ALPL overexpression on A549 (a) and HCC827 (c) cells, Scale bars: 100 µm. b, d The migration and invasion rate of A549 (a) and HCC827 (c) cells, n = 3. e A tail vein injection model was used to evaluate lung metastasis. The front and back images of the lungs are shown, n = 9. f Representative H&E images of A549 (Vector) and A549 (ALPL) tumors, Scale bars: 250 µm, 100 µm or 25 µm. g Quantification of the number of metastatic lung tumors in five pairs of nude mice. h Tube formation assay were performed to determine the effect of ALPL overexpression on the angiogenesis activity of A549 and HCC827, Scale bars: 100 µm. i Capillary length was calculated in A549 and HCC827 cell culture medium. Data were presented as the mean ± SD and analyzed by Student′s t-test. Asterisks (*) represent statistical significance (P < 0.05)

Fig. 4

Alkaline phosphatase (ALPL) inhibits the migration and invasion of LUAD cells by downregulating RhoA protein expression. a Western blot analysis of cell lysates from the indicated cells. b, e Western blot analysis of GFP-RhoA and RhoA expression in A549 (ALPL) and HCC827 (ALPL) cells stably transfected with GFP-RhoA or a vector control plasmid. GAPDH was used as a loading control. Transwell assays were performed to determine the effect of RhoA overexpression on A549 (ALPL) (c) and HCC827 (ALPL) (f) cells, Scale bars: 100 µm. d, g The migration and invasion rate of A549 (ALPL) and HCC827 (ALPL) cells. Data were presented as the mean ± SD and analyzed by Student′s t-test, n = 3. Asterisks (*) indicate a significant increase relative to control vector cells (P < 0.05)

Fig. 5

Alkaline phosphatase (ALPL) inhibits RhoA mRNA transcription by downregulating c-Myc. a Semi-quantitative PCR was used to evaluate RhoA mRNA expression after ALPL overexpression in A549 and HCC827 cells. b RhoA mRNA expression was evaluated by qPCR after ALPL overexpression in A549 and HCC827 cells. c Dual-luciferase reporter assays were used to detect RhoA promoter activity after ALPL overexpression in A549 and HCC827 cells. d TFANSFAC Transcription Factor Binding Sites Software (Biological Database, Wolfenbuttel, Germany) was used for bioinformatics analysis of the RhoA promoter region. Transcription factors for which potentially conserved binding sites were identified in the RhoA promoter region included c-Myc, c-Jun, Foxp3, and Sp1. e Western blot analysis of cell lysates from the indicated cells. GAPDH was used as an internal control. f Western blot analysis of c-Myc and RhoA expression in A549 (ALPL) cells stably transfected with c-Myc or a control plasmid. GAPDH was used as a loading control. g The RhoA mRNA expression level was evaluated by qPCR after overexpression of c-Myc in A549 (ALPL) cells. h Dual-luciferase reporter assays of the RhoA promoter activity after overexpression of c-Myc in A549 (ALPL) cells. i Transwell assays to determine the effect of overexpression of c-Myc on A549 (ALPL) cells, Scale bars: 100 µm. j The migration and invasion rate of A549 (ALPL) cells. All results were presented as the mean ± SD and analyzed by Student’s t-test, n = 3. Asterisks (*) represent statistical significance (P < 0.05)

Fig. 6

Alkaline phosphatase (ALPL) promotes the degradation of c-Myc by activating ERK. a qPCR analysis of the levels of c-Myc mRNA in A549 and HCC827 cells overexpressing ALPL. ns, not significant. b The rate of c-Myc protein degradation in A549 (Vector) and A549 (ALPL) cells. GAPDH was used as an internal control. c Western blot analysis of the levels of c-Myc and p-c-Myc (Thr58/Ser62) in A549 (Vector) and A549 (ALPL) cells after treatment with MG132. d Western blot analysis of the indicated cells for levels of the ubiquitin-related molecules ERK1/2, GSK-3β, p-ERK1/2, and p-GSK-3α/β. GAPDH was used as an internal control. e Western blot analysis of the levels of p-ERK1/2, ERK1/2, c-Myc, and p-c-Myc (Thr58/Ser62) in A549 (ALPL) cells after treatment with PD98059 (100 μM)

Fig. 7

The expression of ALPL, p-ERK, c-Myc and RhoA were determined by IHC staining in the metastatic lung tumors tissues. a Representative IHC images showing the expression of ALPL, p-ERK, c-Myc and RhoA in metastatic lung tumors tissues, n = 9, Scale bars: 400 µm or 100 µm. Protein expression levels of p-ERK (b), c-Myc (c), and RhoA (d) were analyzed by calculating the integrated optical density per stained area (IOD/area) using Image-Pro Plus version 6.0, and the correlation between p-ERK/c-Myc/RhoA and ALPL was analyzed by Pearson’s Chi-square test

Fig. 8

Schematic diagram of the pathway by which ALPL downregulates RhoA via c-Myc. ① ALPL leads to an accumulation of phosphorylated (p)-ERK. ② p-ERK promotes c-Myc phosphorylation at Thr58 and Ser62 which is then degradated through ubiquitin–proteasome system. ③ The reduction of c-Myc leads to a decrease in RhoA transcription, which eventually suppresses LUAD cell metastasis. The dotted line indicates a multi-process, and the solid line indicates a single process