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, 4 (11), e928

MiRNA-296-3p-ICAM-1 Axis Promotes Metastasis of Prostate Cancer by Possible Enhancing Survival of Natural Killer Cell-Resistant Circulating Tumour Cells

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MiRNA-296-3p-ICAM-1 Axis Promotes Metastasis of Prostate Cancer by Possible Enhancing Survival of Natural Killer Cell-Resistant Circulating Tumour Cells

X Liu et al. Cell Death Dis.

Abstract

Natural killer (NK) cells are important in host to eliminate circulating tumour cells (CTCs) in turn preventing the development of tumour cells into metastasis but the mechanisms are very poorly defined. Here we find that the expression level of miR-296-3p is much lower in the non-metastatic human prostate cancer (PCa) cell line P69 than that in the highly metastatic cell line M12, which is derived from P69. We demonstrate that miR-296-3p directly targets and inhibits the expression of intercellular adhesion molecule 1 (ICAM-1) in the malignant M12. The data from clinical tissue microarrays also show that miR-296-3p is frequently upregulated and ICAM-1 is reversely downregulated in PCa. Interestingly, ectopic expression of miR-296-3p in P69 increases the tolerance to NK cells whereas knockdown of miR-296-3p in M12 reduces the resistance to NK cells, which both phenotypes can be rescued by re-expression or silencing of ICAM-1 in P69 and M12, respectively. These results are also manifested in vivo by the decrease in the incidence of pulmonary tumour metastasis exhibited by knockdown of miR-296-3p in M12 when injected into athymic nude mice via tail vein, and consistently down-expression of ICAM-1 reverses this to increase extravasation of CTCs into lungs. Above results suggest that this newly identified miR-296-3p-ICAM-1 axis has a pivotal role in mediating PCa metastasis by possible enhancing survival of NK cell-resistant CTC. Our findings provide novel potential targets for PCa therapy and prognosis.

Figures

Figure 1
Figure 1
Morphological and metastatic differences between P69 and M12. (a) Migration kinetics of P69 and M12, as shown by real-time monitoring of live cell migration (P69-red, M12-green). (b) Light microscopy images of P69 and M12 were taken from cultures grown in 3D culture matrix. Magnification, × 20. (c) Immunofluorescence staining of P69 and M12 grown in 3D Culture Matrix (Vimentin-red, E-cadherin-green, DAPI-blue). (d) The expression levels of E-cadherin in P69 (green line) and M12 (blue line) were detected by flow cytometry. IgG isotype antibody was used as a negative control
Figure 2
Figure 2
P69 are more susceptible to NK cells than M12. (a) Human NK cell cytotoxicity to P69, M12 and K562 (as a positive control) was assessed by calcein release assays in various E/T ratios as described in Materials and Methods section. Data are the mean±S.E.M. of three independent experiments. (b) The mixtures of NK cells and P69 or M12 at an E/T ratio of 1:2 were incubated at 37 °C for 2 h and then stained for CD107α and CD56. The gated on CD56-posivtive cells were analyzed and the numbers in boxes indicated percentage of CD107α-positive NK cells. The data are representative of three separate experiments (left graph). Data were the mean±S.E.M. of three independent experiments (right panel). (c) Polarization of perforin-containing granules in NK cells against P69 or M12 were expressed as percentage of NK cells showing polarized perforin. Data are the mean±S.E.M. from three independent experiments. (d) The conjugate formation of NK cells with P69 or M12 at indicated time points was identified by flow cytometry. The gated on CD56-posivtive cells were analyzed and the numbers in different quadrants indicated percentage of cells. One representative experiment out of three was shown (left graph). The fraction of two-colour NK cells was analyzed and data are the mean±S.E.M. of three independent experiments (right panel). (e) P69 (left panel) or M12 (right panel) was supplemented by the media harvested from 48-h cultural supernatants of P69 or M12 cultures and then calcein release assay was performed at an E/T ratio of 5–1. Data are the mean±S.E.M. of three independent experiments
Figure 3
Figure 3
ICAM-1 contributes to the differential susceptibility of P69 and M12 to NK cells. (a) The expression levels of ligands on P69 (green lines) or M12 (blue lines) paired to NK cell receptors were analyzed by flow cytometer. Red and orange lines represent P69 and M12 with PE or APC-conjugated isotype matched IgG as negative controls, respectively. (b) The NK cell cytotoxicities to ICAM-1-knockdown P69 (P69 shICAM-1) and ICAM-1-overexpressing M12 (M12 ICAM-1) were assessed by calcein release assay. (c) The conjugation of NK cell to P69 shICAM-1 and M12 ICAM-1 were determined. Data are the mean±S.E.M. from three independent experiments. (d) The degranulation of NK cells against P69 shICAM-1 and M12 ICAM-1 was performed. Data are the mean±S.E.M. of three independent experiments. (e) NK cells were incubated with anti-CD11α (a subunit of LFA-1) mAbs to block function of LFA-1 or isotype-matched IgG as a control for 30 min and then mixed with P69 or M12 at an E/T ratio of 5–1 for calcein release assay. Data are the mean±S.E.M. of three independent experiments
Figure 4
Figure 4
MiR-296-3p directly regulates ICAM-1 in PCa cells. (a) Screening of candidate miRNAs (in target cells) involved in the resistance to NK cells were performed using calcein release assay. Data are the mean±S.E.M. of three independent experiments. (b) The expression level of miR-296-3p and transcription level of ICAM-1 in P69 and M12 were analyzed by miRNA transcriptome sequencing and DGE sequencing, respectively. TPM means transcripts per million. (c) Real-time PCR in ΔΔCt method was performed to identify the expression levels of miR-296-3p in P69 and M12. One representative experiment out of three was shown. (d) The protein levels of ICAM-1 in P69 and M12 were determined by western blotting. One representative experiment out of three was shown. (e) The predicted miR-296-3p binding site (red) and their mutant (blue) in the 3′-UTR of ICAM-1 mRNA are indicated. Number shows position of nucleotides in the 3′-UTR of ICAM-1 mRNA. (f) HEK-293T cells were transfected with psiCHECK-2 containing WT or mutant ICAM-1 3′-UTR and miR-296-3p or mock control vector. The Renilla luciferase activity was normalized on the constitutive activity of firefly luciferase. Data are the mean±S.E.M. of three independent experiments. (g) The expression of membrane-bound or total ICAM-1 in P69 and M12 were analyzed by flow cytometry (upper panel) and western blotting (bottom panel), respectively. One representative experiment out of three was shown
Figure 5
Figure 5
MiR-296-3p is upregulated and negatively correlated with ICAM-1 in human PCa tissues. (a) The expression levels of miR-296-3p in human prostate normal tissues (upper panel) and cancer tissues (bottom panel) were detected by ISH as described in Material and methods section. Representative staining tissue sections were presented and the numbers shown in images indicated the ratio of positive staining area. (b) Vertical coordinates indicated the percentage ratio of positive staining area to total tissue area and specimens were ranked into as high (upper 30%), middle (a range of 10%–30%) and low (below 10%) expression levels. Data were analyzed with the Mann–Whitney test to determine the statistic significance (P<0.0001). (c) Human PCa tissue arrays were stained with DIG-conjugated probe for miR-296-3p (left column) or anti-ICAM-1 mAbs (right column). Representative staining tissue sections were presented and the numbers shown in images indicated the ratio of positive staining area to total tissue area. (d) The expression correlation between miR-296-3p and ICAM-1 in PCa tissue was analyzed with the method of Spearman, the coefficient values: r=−0.4365, P=0.0055, n=39
Figure 6
Figure 6
MiR-296-3p affects the sensitivity of PCa cells to NK cells. (a) NK cells were incubated with anti-CD11α mAbs or isotype-matched IgG and then the cytotoxicity assay was performed against miR-296-3p-overexpressing P69 (P69 miR-296-3p) (left panel) and miR-296-3p-silencing M12 (M12 anti-miR-296-3p) (right panel). (b) The NK cell cytotoxicity (left upper panel), degranulation (right upper panel), perforin polarization (left bottom panel) and conjugate formation (right bottom panel) against miR-296-3p P69 and the ICAM-1-overexpressing miR-296-3p P69 (miR-296-3p+ICAM-1 P69) cells were performed as indicated. (c) The NK cell cytotoxicity (left upper panel), degranulation (right upper panel), perforin polarization (left bottom panel) and conjugate formation (right bottom panel) against the anti-miR-296-3p M12 and the ICAM-1-knockdown anti-miR-296-3p M12 (anti-miR-296-3p+shICAM-1 M12) cells were performed as indicated. (d) The conjugate formation between NK cells and P69 (left panel) or M12 (right panel) stable cell lines after blockade of CD11α on NK cells with anti-CD11α mAbs (isotype-matched IgG as a control) were examined as previously described. All above data were the mean±S.E.M. from three independent experiments
Figure 7
Figure 7
MiR-296-3p-ICAM-1-NK signalling axis in control of pulmonary metastasis of M12 in vivo. (a) BALB/c athymic mice (nu/nu) at 8 weeks old were injected intravenously with 2.5 × 106 of M12luc. The transferred M12luc in lungs was detected by IVIS spectrum image system at 0, 24 and 48 h after injection and presented visually with various colour spots based on different intensity of luminescence. (b) The relative amounts of transferred M12luc in lung were presented as percentage. The photon flux of M12 control cells at 0 h was set as a common denominator, by which all other photon flux values of M12 variants at three time points were divided. The data were representative of two independent experiments (five nude mice per group) and expressed as mean±S.E.M. (c) BALB/c athymic mice (nu/nu) at 8 weeks old were injected intraperitoneally with 50 μl of NK cell-depleting anti-asialo GM1 antibody 1 day before intravenous injection of 2.5 × 106 of M12luc. At 24 h after injection of M12luc, 2.5 × 107 of expanded human NK cells were injected intravenously into nude mice. The transferred M12luc in lungs were detected by IVIS spectrum image system at 0, 24 and 48 h, and (d) the data were processed and presented as previously described above
Figure 7
Figure 7
MiR-296-3p-ICAM-1-NK signalling axis in control of pulmonary metastasis of M12 in vivo. (a) BALB/c athymic mice (nu/nu) at 8 weeks old were injected intravenously with 2.5 × 106 of M12luc. The transferred M12luc in lungs was detected by IVIS spectrum image system at 0, 24 and 48 h after injection and presented visually with various colour spots based on different intensity of luminescence. (b) The relative amounts of transferred M12luc in lung were presented as percentage. The photon flux of M12 control cells at 0 h was set as a common denominator, by which all other photon flux values of M12 variants at three time points were divided. The data were representative of two independent experiments (five nude mice per group) and expressed as mean±S.E.M. (c) BALB/c athymic mice (nu/nu) at 8 weeks old were injected intraperitoneally with 50 μl of NK cell-depleting anti-asialo GM1 antibody 1 day before intravenous injection of 2.5 × 106 of M12luc. At 24 h after injection of M12luc, 2.5 × 107 of expanded human NK cells were injected intravenously into nude mice. The transferred M12luc in lungs were detected by IVIS spectrum image system at 0, 24 and 48 h, and (d) the data were processed and presented as previously described above
Figure 8
Figure 8
MiR-296-3p-ICAM-1-NK signalling axis affects survival of M12 in peripheral blood. (a) Quantitative analysis of the residual GFP-positive M12 in peripheral blood of nude mice treated with normal rabbit serum. The cells gated on FSC-SSC plot were analyzed (left upper panel) and the number in oval represented percentage of GFP+/7-AAD viable M12 variant cells (left lower panel). The percentage of GFP+ M12 variants at three time points of 0, 24 and 36 h was normalized by being divided by percentage of GFP+ cells in M12 control cells at 0 h. The data are representative of two independent experiments (n=5) expressed as mean±S.E.M. (right graph). (b and c) Mice were intraperitoneally pre-treated anti-asialo GM1 antibody 1 day before intravenous inoculation of M12 variant cells. Quantitative analysis of residual GFP+ M12 variants in peripheral blood of these mice transferred without (b) or with (c) human NK cells at 24 h after intravenous injection of tumour cells. The data were processed and presented as previously described
Figure 9
Figure 9
Schematic model of miR-296-3p-ICAM-1 axis promotes PCa metastasis by possible enhancing survival of NK cell-resistant CTC. During the early stage of EMT in the long-term malignant transition process from non-metastatic P69 to highly metastatic M12 in nude mice, some of tumour cells acquired genetic changes leading to highly expressing miR-296-3p that directly targeted ICAM-1 gene. These heterogenous tumour cells might invade locally and afterward intravasate into blood vessels to form CTC. Compared with NKs-CTC or parental P69 cells, NKr-CTC with low expression of ICAM-1 on cell surfaces inhibited by miR-296-3p were hardly destroyed by NK cells in blood vessels. The survived NKr-CTC might successively complete the remaining events of the invasion-metastasis cascade including extravasation, micrometastasis, MET and metastatic colonization. CTC, circulating tumour cell; EMT, epithelial–mesenchymal transition; NKs-CTC, natural killer cell-sensitive circulating tumour cell; NKr-CTC, natural killer cell-resistant circulating tumour cells; MET, mesenchymal–epithelial transition

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