Phosphoglucose isomerase/autocrine motility factor mediates epithelial-mesenchymal transition regulated by miR-200 in breast cancer cells

Abstract

Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) plays an important role in glycolysis and gluconeogenesis and is associated with invasion and metastasis of cancer cells. We have previously shown its role in the induction of epithelial-mesenchymal transition (EMT) in breast cancer cells, which led to increased aggressiveness; however, the molecular mechanism by which PGI/AMF regulates EMT is not known. Here we show, for the first time, that PGI/AMF overexpression led to an increase in the DNA-binding activity of NF-κB, which, in turn, led to increased expression of ZEB1/ZEB2. The microRNA-200s (miR-200s) miR-200a, miR-200b, and miR-200c are known to negatively regulate the expression of ZEB1/ZEB2, and we found that the expression of miR-200s was lost in PGI/AMF overexpressing MCF-10A cells and in highly invasive MDA-MB-231 cells, which was consistent with increased expression of ZEB1/ZEB2. Moreover, silencing of PGI/AMF expression in MDA-MB-231 cells led to overexpression of miR-200s, which was associated with reversal of EMT phenotype (i.e., mesenchymal-epithelial transition), and these findings were consistent with alterations in the relative expression of epithelial (E-cadherin) and mesenchymal (vimentin, ZEB1, ZEB2) markers and decreased aggressiveness as judged by clonogenic, motility, and invasion assays. Moreover, either reexpression of miR-200 or silencing of PGI/AMF suppressed pulmonary metastases of MDA-MB-231 cells in vivo, and anti-miR-200 treatment in vivo resulted in increased metastases. Collectively, these results suggest a role of miR-200s in PGI/AMF-induced EMT and thus approaches for upregulation of miR-200s could be a novel therapeutic strategy for the treatment of highly invasive breast cancer.

Figure 1

Effect of PGI/AMF expression on markers of EMT. Expression of E-cadherin, vimentin, ZEB1, ZEB2 was evaluated by Real-Time RT-PCR in (A) MCF-10A and MDA-MB-231 cells, and (B) BT-549 cells. (C) Effect of PGI/AMF expression on DNA-binding activity of NF-κB. Nuclear proteins were subjected to gel shift assay and densitometric analysis of principal NF-κB bands (marked by arrows) is represented in lower panel. Intensity of NF-κB band in the control cells was taken as 1 and the relative intensities of PGI/AMF-transfected/silenced cells are plotted. (D) Effect of PGI/AMF expression on NF-κB target genes, uPAR and MMP-9 was evaluated by western blot analysis. β-actin protein was used as protein loading control. V, vector control; P/A, PGI/AMF-transfected cells; sP/A, PGI/AMF-silenced cells. **p<0.01 vs. vector control.

Figure 2

Re-expression of miR200s reverses EMT induction by PGI/AMF in MCF-10A cells. (A) Basal expression of miR-200a, miR-200b and miR-200c was evaluated by Real-Time RT-PCR in MCF-10A (vector control vs. PGI/AMF expressing cells ± pre-miRNAs). (B-E) Effect of re-expression of miR200s (miR-200a+miR-200b+miR200c) in PGI/AMF expressing MCF-10A cells on the expression of indicated EMT markers was also evaluated by Real-Time RT-PCR. N, non-specific pre-miRNA; PM or M, specific pre-miRNAs (pre-miR200a + pre-miR200b + pre-miR200c) *p<0.05 and **p<0.01 vs. vector / non-specific control.

Figure 3

Suppression of miR200s negates PGI/AMF-silencing-induced effects on EMT in MDA-MB-231 cells. (A) Basal expression of miR-200a, miR-200b and miR-200c was evaluated by Real-Time RT-PCR in MDA-MB-231 (vector control vs. PGI/AMF-silenced cells ± anti-miRNAs). (B-E) Effect of suppression of miR200s (miR-200a+miR-200b+miR200c) in PGI/AMF-silenced MDA-MB-231 cells on the expression of EMT markers was also evaluated by Real-Time RT-PCR. N, non-specific anti-miRNA; AM or M, specific anti-miRNAs (anti-miR200a + anti-miR200b + anti-miR200c) **p<0.01 vs. vector / non-specific control.

Figure 4

miR-200s influence motility and clonogenicity of breast cells. (A) MCF-10A cells and (B) MDA-MB-231 cells were wounded (time 0h) and maintained for 24h in normal medium. (C) Anchorage-dependent assays for clonogenicity were performed as described under “Methods” section. Bar graphs on the right represent quantification of results presented on the left, in each case. V, vector control; P/A, PGI/AMF-transfected cells; sP/A, PGI/AMF-silenced cells, NP, non-specific pre-miRNA; PM, specific pre-miRNAs (pre-miR200a + pre-miR200b + pre-miR200c); NA, non-specific anti-miRNA; AM, specific anti-miRNAs (anti-miR200a + anti-miR200b + anti-miR200c). *p<0.05 and **p<0.01 vs. non-specific control, NS, non-significant.

Figure 5

miR-200s modulate PGI/AMF-mediated EMT and invasion of breast cells. (A) Western blot analysis for the expression of indicated EMT markers. β-actin protein was used as protein loading control. PM, specific pre-miRNAs (pre-miR200a + pre-miR200b + pre-miR200c); AM, specific anti-miRNAs (anti-miR200a + anti-miR200b + anti-miR200c); P/A, PGI/AMF-transfected cells; sP/A, PGI/AMF-silenced cells. (B) Invasion of breast cells MCF-10A (upper panel) and MDA-MB-231 (lower panel) was assayed by plating cells in matrigel-coated inserts. The cells that invaded through the matrigel were stained and photographed using fluorescence microscope. a, MCF-10A control cells; b, PGI/AMF- transfected MCF-10A cells; c, PGI/AMF- transfected MCF-10A cells + pre-miR200s (pre-miR200a + pre-miR200b + pre-miR200c); d, MDA-MB-231 control cells; e, PGI/AMF-silenced MDA-MB-231 cells and f, PGI/AMF-silenced MDA-MB-231 cells + anti-miR200s (anti-miR200a + anti-miR200b + anti-miR200c).

Figure 6

Silencing of PGI/AMF suppresses lung metastasis and anti-miR-200 treatment abrogates this effect. (A) MDA-MB-231 cells (2 × 10⁶ in a total volume of 0.1 ml) were injected via tail vein of ICR-SCID mice, and spontaneous pulmonary metastases were assessed after 5 weeks. The inserts on top of the box plot are representative lungs (left half piece) from each group with arrows indicating grossly visible tumors. (B) H&E staining revealed the extent of metastatic lesions in lungs. V, vector control; sP/A, PGI/AMF-silenced cells, NP, non-specific pre-miRNA; PM, specific pre-miRNAs (pre-miR200s); NA, non-specific anti-miRNA; AM, specific anti-miRNA (anti-miR200b). n=6 mice for each group.

Figure 7

Schematic representation of regulation of EMT and invasion of breast cancer cells by PGI/AMF. The miR200s seem to play a key role in the induction of EMT by PGI/AMF. The complex relationship between miR200s, NF-κB, ZEB1/ZEB2 and E-cadherin, and their regulation by PGI/AMF might be crucial to the acquisition of EMT and aggressive behavior of breast cancer cells.