Apelin Promotes Prostate Cancer Metastasis by Downregulating TIMP2 via Increases in miR-106a-5p Expression

Abstract

Prostate cancer commonly affects the urinary tract of men and metastatic prostate cancer has a very low survival rate. Apelin belongs to the family of adipokines and is associated with cancer development and metastasis. However, the effects of apelin in prostate cancer metastasis is undetermined. Analysis of the database revealed a positive correlation between apelin level with the progression and metastasis of prostate cancer patients. Apelin treatment facilitates cell migration and invasion through inhibiting tissue inhibitor of metalloproteinase 2 (TIMP2) expression. The increasing miR-106a-5p synthesis via c-Src/PI3K/Akt signaling pathway is controlled in apelin-regulated TIMP2 production and cell motility. Importantly, apelin blockade inhibits prostate cancer metastasis in the orthotopic mouse model. Thus, apelin is a promising therapeutic target for curing metastatic prostate cancer.

Keywords: TIMP2; apelin; metastasis; miR-106-5p; prostate cancer.

Figure 1

Apelin levels are associated with clinicopathological characteristics in prostate cancer metastasis. (A,B) Apelin mRNA levels in prostate cancer tissues were examined by the TCGA for tumor volume, lymph node metastasis and Gleason scores. Dots of bar graph means the number of patient. (C) Apelin levels in the prostate gland and cancerous prostate tissue from the Oncomine database. (D) Apelin expression in prostate cancer tissue samples and normal healthy tissue from the GEPIA database. (E) Representative images of IHC staining for apelin in tissue samples from healthy individuals and prostate cancer patients. (F) Increasingly lower survival time was associated with increasing levels of apelin expression. (G) Metastatic prostate cancer exhibited higher levels of apelin expression compared to primary prostate cancer (exposed by AR amplification). Scale bar for all images is 100 μm. NS, no significant difference.

Figure 2

Apelin facilitates the migration and invasion of prostate cancer cells. (A,B) Prostate cancer cells were treated with apelin for 24 h, then migration (n = 4) and invasion (n = 4) was performed by the Transwell assay. (C) Apelin levels in indicated cells were investigated by Western blot (n = 3). (D–F) LNCaP cells were transfected with apelin cDNA, then cell migration (n = 4), invasion (n = 4) and levels of apelin synthesis (n = 3) were performed. (G–I) PC3 cells were transfected with apelin shRNA, then cell migration (n = 4), invasion (n = 4) and apelin expression (n = 3) were performed. * p < 0.05 compared with the control group. Scale bar for all images is 250 μm. Dots of bar graph means the repeat number.

Figure 3

Apelin promotes prostate cancer motility by inhibiting TIMP2 expression. (A) PC3 cells were treated with apelin, then TIMP expression was examined by qPCR (n = 4). (B,C) Cells were treated with apelin for 24 h, then TIMP2 expression was examined by qPCR (n = 4) and Western blot (n = 3). (D–F) Cells were transfected with TIMP2 cDNA followed by stimulation with apelin, then cell migration (n = 4), invasion (n = 4) and levels of TIMP2 expression (n = 3) were examined. (G) Representative images of IHC staining for TIMP2 in tissue samples from healthy individuals and prostate cancer patients. (H,I) Tissue samples from the TCGA and GEPIA databases were measured for levels of TIMP2 in normal and prostate cancer tissue samples. Dots of bar graph means the number of patient. * p < 0.05 compared with the control group; # p < 0.05 compared with the apelin-treated group. Scale bar for all images is 100 μm.

Figure 4

Apelin decreases TIMP2 expression and promotes prostate cancer cell motility by increasing miR-106a-5p expression. (A) MiRNA interference with TIMP2 transcription was predicted by combining 12 datasets from both miRwalk and ONCO.IO. (B,C) Cells were treated with apelin, then miRNA expression was examined by qPCR (n = 4). (D–F) Cells were applied with the miR-106a-5p inhibitor followed by stimulation with apelin, then cell migration (n = 4), invasion (n = 4) and levels of TIMP2 expression (n = 3) were performed. (G) Schematic 3′-UTR representation of the TIMP2 sequence. Red line indicated the point mutation site of miR-105a binding to TIMP2 sequence. (H) PC3 cells were applied with indicated plasmid, then treated with apelin and luciferase activity (n = 3) was measured. (I) Tissue samples from the TCGA database were measured for levels of miR-106a-5p expression in normal and prostate cancer tissues. Dots of bar graph means the number of patient. * p < 0.05 compared with the control group; # p < 0.05 compared with the apelin-treated group.

Figure 5

The c-Src pathway is mediated in apelin-induced migration and invasion of prostate cancer cells. (A–F,H,I) Cells were applied with the c-Src inhibitor (PP2; 1 μM) or siRNA, then incubated with apelin, before examining cell migration (n = 4) and invasion (n = 4), TIMP-2 and miR-106a-5p expression (n = 4). (G) PC3 cells were treated with apelin, then c-Src phosphorylation was investigated by Western blot (up-panel) and the quantify data were provide in low panel (n = 3). * p < 0.05 compared with the control group; # p < 0.05 compared with the apelin-treated group. Dots of bar graph means the repeat number of Western blot assay.

Figure 6

The PI3K/Akt pathway is involved in apelin-induced motility of prostate cancer cells. (A–H) Cells were applied with inhibitors of PI3K (LY294002; 2 μM) and Akt (2 μM), or transfected with PI3K and Akt siRNAs, then applied with apelin, before examining cell migration (n = 4) and invasion (n = 4), TIMP2 and miR-106a-5p expression (n = 4). (I–K) PC3 cells were treated with apelin or pretreated with c-Src and PI3K inhibitors then applied with apelin for 30 min, before examining PI3K and Akt phosphorylation by Western blot (n = 3). * p < 0.05 compared with the control group; # p < 0.05 compared with the apelin-treated group. Dots of bar graph means the repeat number of Western blot assay.

Figure 7

Apelin blockade inhibits prostate cancer metastasis in vivo. (A–E) PC3-Luc (n = 6) or PC3/sh-APLN-Luc cells (n = 6) were injected into the ventral prostates of SCID mice. Tumor growth, tumor metastasis to lungs, livers and legs were investigated by IVIS System. (E) Tissue specimens were stained with apelin or TIMP2 antibody and applied to IHC analysis. * p < 0.05 compared with the control group; Scale bar for all images is 100 μm.

Figure 8

Schematic diagram summarizes the mechanisms by which apelin facilitates metastasis in human prostate cancer cells. Apelin suppresses TIMP2 production and subsequently facilitates the metastatic potential of human prostate cancer cells by increasing miR-106a-5p production via the c-Src, PI3K and Akt signaling pathways.