AMP-activated protein kinase promotes epithelial-mesenchymal transition in cancer cells through Twist1 upregulation

ABSTRACT The developmental programme of epithelial-mesenchymal transition (EMT), involving loss of epithelial and acquisition of mesenchymal properties, plays an important role in the invasion-metastasis cascade of cancer cells. In the present study, we show that activation of AMP-activated protein kinase (AMPK) using A769662 led to a concomitant induction of EMT in multiple cancer cell types, as observed by enhanced expression of mesenchymal markers, decrease in epithelial markers, and increase in migration and invasion. In contrast, inhibition or depletion of AMPK led to a reversal of EMT. Importantly, AMPK activity was found to be necessary for the induction of EMT by physiological cues such as hypoxia and TGFβ treatment. Furthermore, AMPK activation increased the expression and nuclear localization of Twist1, an EMT transcription factor. Depletion of Twist1 impaired AMPK-induced EMT phenotypes, suggesting that AMPK might mediate its effects on EMT, at least in part, through Twist1 upregulation. Inhibition or depletion of AMPK also attenuated metastasis. Thus, our data underscore a central role for AMPK in the induction of EMT and in metastasis, suggesting that strategies targeting AMPK might provide novel approaches to curb cancer spread.

(S1B) T47D and MCF7 cells were cultured in the presence of 100 µM AMPK activator (A769662) or DMSO (vehicle control) for 48 hours, followed by immunoblot analysis for the specified proteins, n=4. Band intensities are represented below the relevant panels.
(S1 C, D and E) BT474 cells were cultured in the presence of 100 µM AMPK activator (A769662) or DMSO (vehicle control) for 48 hours. Thereafter, qPCR analysis (C) was undertaken for the specified transcripts, n=3. Parallel dishes were subjected to immunoblot analysis for the specified proteins, n=3 (D), and immunocytochemical analysis for E-cadherin protein (E). Photomicrographs show representative fluorescent images taken at 20X magnification using Olympus 1X71 microscope; Hoechst 33342 was used for nuclear staining. Graph represents relative fluorescence intensity (RFI) measurements normalized to number of nuclei, n=3.
(S1 F and G) A549 (F) and MDA-MB-231 (G) cells were cultured in the presence of 100 μM AMPK activator (A769662), 10 μM AMPK inhibitor (Compound C) or DMSO (vehicle control) for 48 hours, followed by immunoblot analysis for the specified proteins; n=4. Same DMSO treated samples were used as a control for A769662 or Compound C treated samples, but developed with different exposures to capture the differences. Band intensities are represented below the relevant panels.
(S1I) BT474 cells stably expressing scrambled shRNA or AMPKα2 shRNA were harvested and subjected to immunoblot analysis for the specified proteins, n=2.
(S1J and K) A549 cells stably expressing inducible shRNA against AMPKα2 (clone#1) cultured with and without doxycycline for 48 hours, were harvested and subjected to immunoblot analysis for the specified proteins, n=2 (J). Parallel dishes were subjeted to immunocytochemical analsysis for E-cadherin and vimentin protein, n=2 (K). Photomicrographs show representative fluorescent images taken at 20X magnification using Olympus 1X71 microscope; Hoechst 33342 was used for nuclear staining.
(S1L) A549 cells stably expressing inducible shRNA against AMPKα2 (clone#4) cultured with and without doxycycline for 48 hours, were harvested and subjected to immunoblot analysis for the specified proteins, n=2 (S1M and N) MDA-MB-231 cells stably expressing scrambled shRNA or AMPKα2 shRNA were harvested and subjected to immunoblot analysis for the specified proteins, n=3 (K). Parallel dishes were subjected to immunocytochemistry analysis for Vimentin, n=3 (L).
(S1O) MDA-MB-231 cells stably expressing inducible shRNA against AMPKα2 (clone #4) cultured with and without doxycycline for 48 hours, were harvested and subjected to immunoblot analysis for the specified proteins, n=2.
(S1P) MDA-MB-435S cells transfected with control siRNA or AMPKα2 siRNA for 48 hours were harvested and subjected to immunoblot analysis for the specified proteins; n=4.  (S3A) MDA-MB-231 cells stably expressing scrambled shRNA or AMPKα2 shRNA were cultured in the presence of 150 µM CoCl 2 for 48 hours. Thereafter, cells were harvested and immunoblot analysis was undertaken. Graph represents densitometric quantification of the specified proteins normalized to α-Tubulin; error bars represent SEM, n=4.
(S3B) MDA-MB 231 cells were cultured with 150 µM CoCl 2 in the presence or absence of 10 µM AMPK inhibitor (Compound C) for 48 hours. Thereafter, cells were harvested for RNA isolation and subjected to qRT-PCR for specified transcripts. Graph represents fold change in gene expression normalized to β2m; error bars represent SEM, n=3.
(S3C) A549 cells were cultured with 5 ng/ml TGFβ in the presence or absence of 10 µM AMPK inhibitor (Compound C) for 24 hours. Thereafter, cells were harvested and immunoblot analysis was undertaken for EMT marker proteins. Band intensities are represented below the relevant panels; n=3.
(S3D) MDA-MB-231 cells were cultured with 5 ng/ml TGFβ in the presence or absence of 10 µM AMPK inhibitor (Compound C) for 24 hours. Thereafter, cells were harvested for RNA isolation and subjected to qRT-PCR for specified transcripts. Graph represents fold change in gene expression normalized to β2m; error bars represent SEM, n=3.
(S3E) MDA-MB-231 cells stably expressing scrambled shRNA or AMPKα2 shRNA were cultured in the presence of 5 ng/ml of TGFβ for 24 hours. Thereafter, the cells were harvested and subjected to immunoblot analysis. Graph represents densitometric quantification of the specified proteins normalized to α-Tubulin; error bars represent SEM, n=3.