AUF1 promotes stemness in human mammary epithelial cells through stabilization of the EMT transcription factors TWIST1 and SNAIL1

Abstract

The AU-rich element RNA-binding protein 1 (AUF1) is an RNA-binding protein, which can both stabilize and destabilize the transcripts of several cancer-related genes. Since epithelial-to-mesenchymal transition (EMT) and the acquisition of cancer stem cell traits are important for cancer onset and progression, we sought to determine the role of AUF1 in these two important processes. We have shown that AUF1 induces EMT and stemness in breast epithelial cells via stabilization of the SNAIL1 and TWIST1 mRNAs, and their consequent upregulation. Indeed, AUF1 binds the transcripts of these two genes at their 3'UTR and reduces their turnover. Ectopic expression of AUF1 also promoted stemness in mammary epithelial cells, and thereby increased the proportion of cancer stem cells. Importantly, breast cancer cells that ectopically express AUF1 were more efficient in forming orthotopic tumor xenografts in nude mice than their corresponding controls with limiting cell inocula. On the other hand, AUF1 downregulation with specific siRNA inhibited EMT and reduced the stemness features in breast cancer cells. Moreover, AUF1 knockdown sensitized breast cancer cells to the killing effect of cisplatin. Together, these findings provide clear evidence that AUF1 is an important inducer of the EMT process through stabilization of SNAIL1 and TWIST1 and the consequent promotion of breast cancer stem cells. Thereby, AUF1 targeted molecules could constitute efficient therapeutics for breast cancer patients.

Fig. 1. Ectopic expression of AUF1 induces EMT in mammary epithelial cells.

a MCF10A and MCF7 cells were infected with lentivirus-based vectors either empty (MCF10A-C) (MCF7-C) or bearing p37^AUF1-ORF (MCF10A-ORF) (MCF7-ORF). Whole-cell lysates were prepared from these cells and were used for immunoblotting using antibodies against the indicated proteins. The numbers bellow the bands represent fold change relative to the corresponding control after correction against the internal controls β-actin or GAPDH. The levels of phosphorylated proteins were normalized against the total amount of their relative non-phosphorylated forms. b Total RNA was purified, and then amplified by qRT-PCR. The relative mRNA expression levels were normalized against GAPDH. Error bars represent means ± SD (*P < 0.05, **P < 0.01). c Representative images of cells. Scale bar, 50 μm. d Exponentially growing cells were added in SFM to the upper wells of the CIM plates either separated by a matrigel basement membrane matrix (Invasion) or without (Migration), and the migration/invasion were assessed for 24 h using the RTCA-DP xCELLigence System. e Exponentially growing cells were added in complete medium to the wells of an electronic microtiter plate (E-plate). The proliferation rate was measured for a period of 72 h.

Fig. 2. AUF1 downregulation inhibits EMT in TNBC cells.

a MDA-MB-231 and BT-20 cells were transfected with control or AUF1-siRNA (MDA-AUF1si/MDA-C) or (BT20-AUF1si/BT20-C), respectively. Whole-cell lysates were prepared and were used for immunoblotting analysis using antibodies against the indicated proteins, and GAPDH was used as an internal control. The numbers bellow the bands represent fold change relative to the corresponding control. b Total RNA was purified from the indicated cells and used for qRT-PCR. Error bars represent means ± SD (*P < 0.05, **P < 0.01). c, d Cell proliferation, migration, and invasion abilities were assessed for the indicated periods of time using the RTCA-DP xCELLigence System.

Fig. 3. AUF1 stabilizes SNAIL1 and TWIST1 mRNAs.

a–d Cells were treated with actinomycin D (5 μg/ml), and then total RNA was extracted at different periods of time, and was subjected to qRT-PCR. Error bars represent means ± SD (*P < 0.05, **P < 0.01). e Sequence alignment of the indicated human mRNA 3′UTR showing the WT and the mutated potential AUF1-binding sites. f Biotinylated 3′UTR for the indicated mRNAs bearing either WT or mutated sequence of the AUF1-binding site was incubated with cytoplasmic cellular lysate from MDA-MB-231 cells expressing the indicated constructs, and the association of AUF1 with these mRNAs was detected by immunoblotting. g MCF7-C and MCF7-ORF cells were stably transfected with the luciferase reporter vector bearing either wild-type TWIST1 and SNAIL1 3′UTR or their mutated sequences as shown in e. The reporter activity was assessed at 48 h post-transfection. Data (mean ± SEM, n = 4) were presented as % change in reporter activity as compared to control cells (**) or to the mutated 3′UTR ($). **P < 0.002 and ^$P < 2 × 10^–5.

Fig. 4. Ectopic expression of AUF1 induces stemness in MCF10A and MCF7 cells.

a Whole-cell lysates were prepared from the indicated cells and were used for immunoblotting using antibodies against the indicated proteins, and GAPDH and β-actin were used as internal controls. The numbers below the bands represent fold change relative to the corresponding control after correction against the internal controls β-actin or GAPDH. b Total RNA was purified from the indicated cells and used for qRT-PCR. Error bars represent means ± SD (*P < 0.05, **P < 0.01). c Immunofluorescence assay for the indicated cells using antibodies against the indicated proteins. Scale bars represent 25 µm. d FACS analysis of CD44^high/CD24^low and ALDH^high stem cell-like subpopulation in the indicated cells. The numbers in the boxes indicate the proportion of cells. e Cells (1000) were cultured in ultra-low attachment 96-well plates in the presence of specific stem cell medium. Left panel, representative images of mammospheres. Scale bar, 50 μm. Right panel, graphs depicting number of formed mammospheres. Experiments were performed in triplicate and several times; error bars represent means ± SD (*P < 0.05). f Cells were plated in soft agar for colony formation. After 2 weeks colonies were photographed using an inverted microscope. g Female nude mice were injected with the indicated number of MCF7-ORF and MCF7-C cells under the right and left nipples, respectively. Five months post-injection, pictures of tumors were taken. Red arrows indicate the grown tumors in mice.

Fig. 5. AUF1-depenent induction of stemness is TWIST1-SNAIL1-related.

a MCF7 cells expressing AUF1-ORF were transfected with plasmids bearing different sequences of either TWIST1-shRNA, SNAIL1-shRNA or a scrambled sequence. After 72 h, cells were harvested and total RNA was purified and used for qRT-PCR. Error bars represent means ± SD. b The migration/invasion as well as proliferation capabilities of the indicated cells were assessed using the RTCA-DP xCELLigence system. c, d Figure legends as in Fig. 4b, e, f. Figure legends as in Fig. 4e, f. Error bars represent means ± SD (*P < 0.05).

Fig. 6. AUF1 downregulation decreases stemness in TNBC cells and enhances their sensitivity to cisplatin.

a MDA-MB-231 cells were transfected with specific AUF1 siRNA (AUF1-siRNA) or a scrambled sequence (CTRL), and then whole-cell lysates were prepared and were used for immunoblotting using antibodies against the indicated proteins. The numbers bellow the bands represent fold change relative to the corresponding control. b Total RNA was purified and was used for qRT-PCR. Error bars represent means ± SD (**P < 0.01). c Exponentially growing cells were treated either with DMSO (control) or with cisplatin (20 and 30 μM) for 72 h. Cell viability was measured using the WST-1 assay. Error bars represent means ± SD (*P < 0.05).